In 1991, HYDRA operatives inside the Russian Armed Forces travel to their Siberian facility and revive the Winter Soldier from a cryogenic state. After using a set of conditioned trigger words to ensure the Winter Soldier's loyalty, Vasily Karpov orders him to intercept an automobile and execute its occupants. In the present, approximately one year after the Ultron Offensive, public opinion of the Avengers has become increasingly divided due to the events in New York, Washington, D.C. and Sokovia.

Captain America, Black Widow, Falcon and Scarlet Witch learn that Brock Rumlow plans to steal a biological chemical weapon from the Institute for Infectious Diseases in Lagos, Nigeria. After being cornered, Rumlow attempts to commit suicide with a bomb vest, trying to take Rogers with him in an act of revenge. Maximoff contains the explosion, but she loses control of her powers and inadvertently destroys a nearby building, killing several relief aid workers from Wakanda.

At MIT, Tony Stark demonstrates B.A.R.F. – a piece of technology aimed to recreate and relive old memories, and follows with a speech where he introduces a grant being awarded to all students, giving them all the proper funding to move forward with their own inventions and ideas. Before leaving the stage, he is shaken when the teleprompter makes reference to Pepper Potts, whom he has broken up with over his refusal to give up being Iron Man.

Outside, he is confronted by Miriam Sharpe whose son was killed in the battle with Ultron in Sokovia, whose story shakes Stark.

One month after the incident in Lagos in their headquarters, the Avengers are visited by Stark and Thaddeus Ross, waiting for them. Ross explains that, due to the events in Lagos, the United Nations has decided they can no longer be allowed to operate privately. To that end, it has been decided that the Avengers will sign the Sokovia Accords, which will establish an international panel to control the Avengers' movements as well as monitor and police the rapidly growing number of enhanced individuals.

The team is divided over the act; Stark supports government oversight because he feels guilty for his role in creating Ultron and the devastation Ultron caused. He also feels that the Avengers need to take more responsibility for the consequences of their actions, as he did when he discovered Stark Industries was secretly shipping weapons to Ten Rings. However, Rogers worries that the Avengers may be compromised if it comes under the control of a nefarious third party, such as the infiltration of S.H.I.E.L.D. by HYDRA. Ross states that refusing to sign the accords will be seen as an act of resignation.

Meanwhile, retired Karpov is ambushed by Helmut Zemo, who asks for information regarding the 1991 mission. Karpov refuses, forcing Zemo to kill him.

During their debate about the Accords, Rogers learns that Peggy Carter has passed away in her sleep and abruptly leaves for her funeral in London. While there, he learns Sharon Carter was Peggy's great-niece as she delivers a eulogy about how one should never back down from what they believe in. This solidifies Roger's resolve not to sign the accords. Wanda finds herself confined to the facility by Vision, ostensibly for her safety since emotions are running high after what happened in Lagos.

In Vienna, where the signing of the Accords is to take place, a terrorist bomb kills King T'Chaka of Wakanda. When security cameras reveal the suspect as Bucky Barnes, T'Chaka's son, T'Challa, vows revenge. Against Romanoff's advice, Rogers and Wilson decide to go behind Ross' back and bring in Barnes themselves. Following a tip-off by Carter, and a grueling chase, Barnes is captured, and Rogers, Wilson, and T'Challa are arrested for obstruction of justice.

While Barnes is in captivity in Berlin, Rogers and Stark argue about the Accords further, with Rogers nearly convinced to sign until he learns of Wanda's in-house arrest. At a power grid station outside the city, a box sent by Zemo contains an e-bomb, which explodes and shuts down electricity for the entire city, including the cameras and computers that we're monitoring on Barnes. Barnes is released by Zemo, who uses HYDRA's trigger words to send him on a rampage. Rogers and Wilson intervene just as Barnes is escaping containment, with Rogers attempting to calm Barnes while Wilson chases after Zemo, attempting to flee the facility. Zemo escapes, and Barnes nearly gets away in a helicopter before being caught by Rogers.

In a panic, he intentionally crashes the chopper, and as it falls into the nearby river, pulls Rogers in with him before being knocked unconscious by the impact. After coming to his senses, Barnes tells Rogers that Zemo is headed for the abandoned HYDRA facility in Siberia where he and five other Winter Soldiers were created using the Super Soldier Serum he stole in 1991. Believing Zemo intends to release the other Winter Soldiers and unleash them on the world, Rogers calls upon Clint Barton for aid in freeing Wanda, which Barton agrees to since he feels he owes her a debt as her brother sacrificed himself to save his life in Sokovia. He manages to break into the New Avengers Facility and convinces Wanda to come with him, who uses her abilities to overpower Vision. Falcon decides to recruit Scott Lang to join the team as well, having been impressed by his abilities following their duel a few months prior. Lang, who is in awe at getting to meet Captain America, quickly and easily agrees to join Rogers.

Back at the Joint Counter Terrorist Centre compound, Stark convinces Ross to give him 36 hours to bring his renegade comrades in and assembles Romanoff, T'Challa, James Rhodes, and Vision. Feeling he is still undermanned, Stark travels to New York City and meets Peter Parker, a high school student who has been using enhanced agility and tensile webbing of his own design to fight crime while disguised as a spider-based hero known as Spider-Man. When Stark asks him why he's doing this, Parker explains that he understands the price of not doing the right thing.

Trying to flee Berlin and reach Siberia before Zemo, Rogers and his team assemble at Flughafen Leipzig-Halle airport, where they hope to take a chopper out of the country. When sirens indicate the airport is being evacuated, Rogers instructs his team to suit up and travels to a chopper alone, where he is confronted by Stark alongside his allies. When Rogers refuses to back down, the Avengers get into a massive fight which destroys a large section of the airport. During the fight, the Avengers decides to buy time for Rogers and Barnes to reach the Quinjet, at the expense of imprisonment. Ant-Man uses his suit to grow gargantuan, which tips the scales and creates enough of a distraction to allow Rogers and Barnes to reach a jet. The two are confronted by Romanoff, but she can't bring herself to take them down and allows them to leave in Stark's Quinjet, while the rest of the Avengers are captured. As Stark and Rhodes chase the jet, Vision attempts to blast Falcon, who is following them, out of the sky. However, he misses and hits Rhodes, who plummets to the ground and is left paralyzed. Romanoff, knowing she will be arrested for acting against the accords, goes into hiding. Parker is injured in the fight, and a grateful Stark sends the boy back home, much to the former's dismay.

Stark finds evidence that Barnes was framed by Zemo, and finds that the rest of the Avengers have been thrown into a top-secret prison called the Raft designed to hold enhanced people. He visits the dismayed former Avengers and temporarily disables security in order to learn from Wilson where Rogers and Barnes are going. He follows the two to Siberia and is shadowed by T'Challa. Rogers and Stark reconcile, and as they explore the facility, they discover that the other Winter Soldiers have been murdered by Zemo who had never intended to release them. Zemo reveals that he is a Sokovian and wants to punish the Avengers for the death of his family during Ultron's attack. Zemo shows Stark a security video from 1991, which reveals Barnes as the assassin who murdered Stark's parents to steal samples of the Super Soldier Serum from their car. Rogers tells Stark that he knew the circumstances of their deaths, though not that it was Barnes himself who killed them. Stark see through the lie and Rogers admits that he knew it was Barnes all along. A disillusioned and enraged Stark then turns on Barnes, forcing Rogers to fight him to save his friend's life. While trying to crush Stark's arc reactor, Barnes' robotic arm gets blasted off by his suit's unibeam.

Rogers finally manages to disable Stark's armor and begins to depart with Barnes. As Rogers leaves, Stark bitterly reminds him that his shield doesn't belong to him, but rather his father, who made it. Accepting that he is no longer worthy of it or the title of Captain America, Rogers drops the shield and leaves with Barnes. Having succeeded in his plans of ripping apart the Avengers, Zemo waits outside where he meets with T'Challa and the two discuss vengeance and the terrible things it can lead men to do. T'Challa has decided he will not let his desire for revenge consume him like it did Zemo and the Avengers and prevents him from committing suicide, content on sending him to prison. Under the custody of Everett Ross, Zemo hints that while he may be imprisoned, his grand plan wasn't as much of a failure as Ross suggests it was, knowing that the Avengers may now be irretrievably fractured.

Back at the Avengers facility, Stark builds an exoskeletal frame for Rhodes, to help him regain the use of his legs. Rhodes assures his friend that, despite what happened, like him, he still believes he made the right choice. Stark receives a package containing a phone and a letter from Rogers. In the letter, Rogers apologizes for everything that happened and for not telling him the truth about his parents, as well as noting his regret that he cannot accept the accords. He tells Stark that he knows the day will come when the team will need to assemble and act like one again. When that day arrives, Rogers and his team he freed from prison will be there.

Several weeks later, Barnes and Rogers are granted asylum in Wakanda by T'Challa. Barnes chooses to undergo cryostasis until his brainwashing can be completely removed or suppressed successfully. As Rogers warns T'Challa that the governments of the world will come to Wakanda if they find out Barnes is there, T'Challa dares them to try.

Shortly after the clash, Parker discovers a portable holographic computer that Stark had programmed into his Web-Shooters.