A fishing trawler finds a man (Damon) floating in the Mediterranean Sea with two gunshot wounds in his back and a device with the number of a Swiss bank account embedded in his hip. Suffering from retrograde amnesia, he finds he is versed in several European languages and can perform basic tasks such as writing and tying knots, but he cannot remember anything about himself or how he got there.
When the ship docks, he sets off for Zurich to investigate the bank account. At CIA headquarters in Langley, Deputy Director Ward Abbott (Cox) finds out about a failed assassination attempt on deposed African dictator Nykwana Wombosi (Akinnuoye-Agbaje). Alexander Conklin (Cooper), the head of the black ops group Operation Treadstone, assures Abbott that he will destroy any evidence connecting them to the field agent responsible for the operation. He activates three operatives to take down the agent: Castel (Nicky Naude), Manheim (Russell Levy) and the Professor (Owen). At the bank in Zurich, the mysterious man opens his safe deposit box to find several passports with his picture, large amounts of money, and a gun. With still no idea what his name is, he assumes the one from the first US passport, Jason Bourne. When American and Swiss authorities attempt to capture him at the US consulate, he gives Marie Helena Kreutz (Potente) $20,000 to take him to Paris, the city on his Jason Bourne passport.
When Bourne arrives at the address on his passport, he is immediately recognized by the building superintendent, and based on the contents of his apartment, concludes he is in the shipping business. In search of more clues, he hits redial on his phone and is connected to the Hotel Regina, who recognize one of his aliases, John Michael Kane. They tell him that Kane was a guest who died two weeks before in a car crash. A few moments later, Castel blasts through a window and engages Bourne in hand-to-hand combat.
After Bourne subdues him he attempts to interrogate him, but Castel jumps out the window, preferring suicide. Marie finds wanted posters in Castel's bag with both her and Bourne's pictures on them. After spending the night, they follow the trail to the Hotel Regina. Meanwhile, Conklin plants a body in the Parisian morgue to fool Wombosi into thinking Kane is dead, but Wombosi knows that the body is not his assailant, and Conklin has Wombosi killed in his home by the Professor. Bourne investigates the incident, the previous assassination attempt, and John Michael Kane's contacts, and concludes that he is the failed killer. He and Marie escape the city into the country to stay at Marie's stepbrother's house, where Jason decides that he no longer wants to know about his past.
In the morning, the Professor comes for Jason, who turns the tables on his attacker, shooting and interrogating him. Bourne finds out that he is Treadstone's lone agent in Paris, and sends Marie away with the rest of the money for her own safety. He uses the Professor's phone to arrange a meeting with Conklin, which he uses as a distraction to plant a tracking device on Conklin's vehicle and discover the location of the CIA's Paris safe house.
When he meets Conklin, he begins to remember his last mission. He had backed out of the Wombosi assassination after seeing his children, and was shot while attempting to escape the boat, left floating in the water. He tells Conklin that he is leaving Treadstone, and after a shoot out, leaves the safe house. Abbott decides that Treadstone should be closed down, and has the last operative take out Conklin. He goes before the Oversight Committee and explains that Treadstone's purpose was simply to serve as an assassin-training program then immediately shifts the focus of the hearing to an idea for a new project codenamed "Blackbriar." Some time later, Jason finds Marie in Mykonos, renting out scooters to tourists, and the two reunite at the film's conclusion.