Following his pursuit by Kirill, Jason Bourne (Matt Damon) eludes Moscow police while wounded through a train station and deals with more flashbacks of when he first joined Operation Treadstone.
Six weeks later, CIA Deputy Director Pamela Landy (Joan Allen) divulges the audiotaped confession of Ward Abbott, the late former head of Treadstone, to Director Ezra Kramer (Scott Glenn). Meanwhile, in Turin, journalist Simon Ross (Paddy Considine) of The Guardian meets an informant to learn about Bourne and Operation Blackbriar, the program succeeding Treadstone. The CIA tracks Ross as he returns to London, after his mention of “Blackbriar” during a cell phone call to his editor is detected by the ECHELON system. Bourne reappears in Paris to inform Martin Kreutz (Daniel Brühl), the brother of his girlfriend Marie Helena Kreutz (Franka Potente), of her assassination in India, also in the previous film.
Bourne reads Ross’s articles and arranges a meeting with him at London Waterloo station. Bourne realizes that the CIA is following Ross and helps him evade capture for a while, but when he panics and ignores Bourne’s instructions, Ross is shot and killed by Blackbriar assassin Paz (Édgar Ramírez) on orders of Deputy Director Noah Vosen (David Strathairn). Vosen’s team, reluctantly assisted by Landy, analyzes Ross’s notes and identifies his source as Neal Daniels (Colin Stinton), a CIA Station chief involved with Treadstone and Blackbriar. Bourne makes his way to Daniels’ office in Madrid but finds it empty. He incapacitates gunmen sent by Vosen and Landy. Nicky Parsons (Julia Stiles), a former Treadstone operative who shares a history with Bourne, tells him that Daniels has fled to Tangier and aids his escape from an arriving CIA unit.
Nicky learns that Blackbriar “asset” Desh Bouksani (Joey Ansah) has been tasked with killing Daniels. Vosen sees that Nicky accessed information about Daniels and sends Bouksani after Nicky and Bourne as well, a decision with which Landy fiercely disagrees. Bourne follows Bouksani to Daniels but fails to prevent Daniels’s death by a planted bomb. However, Bourne manages to kill Bouksani before he can kill Nicky. After sending Nicky into hiding, Bourne examines the contents of Daniels’ briefcase and finds the address of the deep-cover CIA bureau in New York City, where Vosen directs Blackbriar. Bourne travels to New York.
Landy receives a phone call from Bourne, which is intercepted by Vosen. When Landy tells him that his real name is David Webb and gives him the birth date “4-15-71”, Bourne tells Landy to “get some rest” because she looks tired, tipping off that he is in New York and watching her from an overlooking building. Vosen intercepts a text to Landy from Bourne of a location to meet up, and leaves his office with a tactical team. Bourne, however, waits for them all to leave, enters Vosen’s office, and takes classified Blackbriar documents. When he realizes that he has been tricked, Vosen sends Paz after Bourne, but the resulting car chase ends with Bourne forcing Paz’s car to crash into a concrete barrier. Bourne holds the injured Paz at gunpoint, but spares his life.
Bourne arrives at a hospital at 415 East 71st Street, having figured out Landy’s coded message. Outside, Bourne meets Landy and gives her the Blackbriar files before going inside. Vosen also figures out Landy’s code and warns Dr. Albert Hirsch (Albert Finney), who ran Treadstone’s behavior modification program, that Bourne is coming. He follows Landy inside the building but is too late to stop her from faxing the Blackbriar documents out. Meanwhile, Hirsch is confronted by Bourne on an upper floor, who now remembers that he volunteered for Treadstone. As Bourne flees to the roof, he is confronted by Paz, who asks, “Why didn’t you take the shot?” Bourne asks Paz if he knows why he’s supposed to kill him, and repeats the dying words of The Professor in The Bourne Identity: “Look at us. Look at what they make you give.” Paz lowers his gun, but Vosen appears and shoots at Bourne as he jumps into the East River.
Some time later, Nicky watches a news broadcast about the exposure of Operation Blackbriar, the arrests of Hirsch and Vosen, a criminal investigation against Kramer, and the whereabouts of David Webb, a.k.a. Jason Bourne. Upon hearing that his body has not been found after a three-day search of the river, Nicky smiles. Bourne is shown swimming away in the East River.