In 1991, the brainwashed super-soldier Bucky Barnes is dispatched from a Hydra base in Siberia to intercept an automobile carrying a case of super-soldier serum. In the present day, approximately one year after Ultron's defeat in the nation of Sokovia at the hands of the Avengers (Avengers: Age of Ultron),Steve Rogers, Natasha Romanoff, Sam Wilson, and Wanda Maximoff stop Brock Rumlow from stealing a biological weapon from a lab in Lagos. Rumlow blows himself up to avoid capture, and when Maximoff tries to displace the blast into the sky with telekinesis, it destroys a nearby building, killing several Wakandan humanitarian workers.

At the team's headquarters, U.S. Secretary of State Thaddeus Ross informs them that the United Nations (UN) is preparing to pass the Sokovia Accords, which will establish a UN panel to oversee and control the Avengers. The team is divided: Tony Stark supports oversight because he feels responsible for Ultron's creation and Sokovia's destruction, while Rogers has more faith in his own judgment than that of the government. At a conference in Vienna where the accords are to be ratified, a bomb kills King T'Chaka of Wakanda. Security footage indicates the bomber is Barnes, who T'Chaka's son, T'Challa, vows to kill. Informed by Sharon Carter of Barnes' whereabouts and the government's intentions to kill him, Rogers goes to bring in Barnes—his childhood friend and war comrade—himself. Rogers and Wilson track him to Bucharest and attempt to protect him from the authorities and T'Challa, but Rogers, Wilson, Barnes, and T'Challa are arrested. 

Helmut Zemo tracks down and kills Barnes' old Hydra handler, stealing a book containing the trigger words that activate Barnes' brainwashing. Infiltrating the facility where Barnes is held, Zemo recites the words to make Barnes obey him. He questions Barnes, then sends him on a rampage to cover his own escape. Rogers stops Barnes and sneaks him away. When Barnes regains his senses, he explains that Zemo is the real Vienna bomber and wanted the location of the Siberian Hydra base, where other brainwashed super-soldiers are kept in cryogenic stasis. Unwilling to wait for authorization to apprehend Zemo, Rogers and Wilson go rogue, and recruit Maximoff, Clint Barton, and Scott Lang to their cause. With Ross's permission, Stark assembles a team composed of Romanoff, T'Challa, James Rhodes, Vision, and Peter Parker to capture the renegades. Stark's team intercepts Rogers' team at Leipzig/Halle Airport, where they fight until Romanoff allows Rogers and Barnes to escape. The rest of Rogers' team is captured and detained at the Raft prison, while Rhodes is partially paralyzed after being inadvertently shot down by Vision, and Romanoff is forced to go into hiding. 

Stark discovers evidence that Barnes was framed by Zemo and shows this evidence to Wilson, who gives him Rogers' destination. Without informing Ross, Stark goes to the Siberian Hydra facility and strikes a truce with Rogers and Barnes, unaware he was secretly followed by T'Challa. They discover that the other super-soldiers have been killed by Zemo, who shows them footage from Hydra's archives; it reveals that Barnes killed Stark's parents during his mission in 1991. Enraged that Rogers kept this from him, Stark turns on them both, blasting off Barnes' robotic arm. Rogers disables Stark's armor and departs with Barnes, leaving his shield behind. Satisfied that he has avenged his family's death in Sokovia by irreparably fracturing the Avengers, Zemo attempts suicide, but T'Challa stops him and he is taken to the authorities.

In the aftermath, Stark provides Rhodes with exoskeletal leg braces that allow him to walk again, while Rogers breaks his allies out of the Raft. In a mid-credits scene, T'Challa grants asylum to Barnes, who chooses to return to cryogenic sleep until a cure for his brainwashing is found. In a post-credits scene, Parker tests a new gadget that he received from Stark.