In the Mediterranean Sea, Italian fishermen rescue an American adrift with two gunshot wounds in his back. They tend to his wounds and find he has no memory of his identity, but retains his speech, advanced combat skills and fluency in several languages. The skipper finds a laser projector under the man's hip that gives the number of a safe deposit box in Zürich. Upon landing in Imperia, Italy, the American goes to the bank in Switzerland to investigate the deposit box. He finds it contains money in various currencies, passports and identity cards with his picture on them, and a handgun. The man takes everything but the gun, using the name on the American passport, Jason Bourne.
After Bourne's departure, a bank employee contacts Operation Treadstone, a CIA black ops program. Treadstone's head, Conklin, issues alerts to police to capture Bourne and activates three agents to kill him: Castel, Manheim, and the Professor. CIA Deputy Director Abbott contacts Conklin about a failed assassination attempt against exiled African dictator Wombosi. Conklin promises Abbott that he will deal with the Treadstone agent who failed.
Bourne tries evading the Swiss police by using his U.S. passport to enter the American consulate, but is pursued by Marine guards. He escapes the consulate, offering a German woman, Marie Kreutz, $20,000 to drive him to an address in Paris listed on his French driving license. At the address, an apartment, he hits redial on the phone and reaches a hotel. He inquires about the names on his passports there, learning that a "John Michael Kane" was registered but died two weeks prior in a car crash. Castel ambushes Bourne and Marie in the apartment, but Bourne gets the upper hand. Instead of allowing himself to be interrogated, Castel throws himself from a window to his death. Among Castel's belongings, Marie finds wanted posters of Bourne and herself, and agrees to help him. After Bourne evades Paris police in Marie's car, the two spend the night in a Paris hotel.
Wombosi obsesses over the attempt on his life. Conklin, having anticipated this, planted a body under the name "John Michael Kane" in Hoffenmein morgue, Paris to appear as the assailant, but Wombosi is not fooled and threatens to report CIA secrets to the media. The Professor assassinates Wombosi on Conklin's orders. Bourne, posing as Kane, learns about Wombosi's yacht, and that the assailant was shot twice in the back during the escape; he realizes he was the assailant. He and Marie take refuge at the French countryside home of Marie's friend Eamon and his children. Under pressure from Abbott to tie off the Wombosi matter, Conklin tracks Bourne's location and sends the Professor there, but Bourne shoots him with Eamon's shotgun, mortally wounding him. The Professor reveals their shared connection to Treadstone before dying. Bourne sends Marie, Eamon, and Eamon's children away for their protection, then contacts Conklin via the Professor's phone. Conklin agrees to meet Bourne, alone, in Paris. When Bourne sees Conklin has not come alone he abandons the meeting, but places a tracking device on Conklin's car, leading Bourne to Treadstone's safe house in Paris.
Bourne breaks in and holds Conklin and logistics technician Nicolette "Nicky" Parsons at gunpoint. Conklin reveals to Bourne his association with Treadstone and presses him to remember his past. Bourne recalls his attempt to assassinate Wombosi through successive flashbacks. As Kane, and working under orders from Treadstone, Bourne infiltrated Wombosi's yacht and got close enough to assassinate him. However, Bourne was unable to find the nerve to kill Wombosi while his children were present, and instead fled, being shot during his escape. Bourne announces he is resigning from Treadstone and warns Conklin not to follow him. As agents descend on the safe house, Bourne fights his way free. When Conklin leaves the safe house, he encounters Manheim, who kills him under Abbott's orders. Abbott then shuts down Treadstone.
Abbott reports to an oversight committee that Treadstone is "all but decommissioned" before discussion turns to a new project codenamed "Blackbriar". Bourne finds Marie renting out scooters to tourists on Mykonos, and the two reunite.
Matt Damon as Jason Bourne
Franka Potente as Marie Kreutz
Chris Cooper as Alexander Conklin
Clive Owen as The Professor
Brian Cox as Ward Abbott
Adewale Akinnuoye-Agbaje as Nykwana Wombosi
Gabriel Mann as Danny Zorn
Julia Stiles as Nicky Parsons
Orso Maria Guerrini as Giancarlo
Tim Dutton as Eamon
Nicky Naude as Castel
Russell Levy as Manheim
Vincent Franklin as Rawlins
Walton Goggins, Josh Hamilton, and Brian Huskey appear as Treadstone research technicians. David Bamber has a minor role as a clerk at the American consulate who denies Marie a student visa, David Gasman has one as the deputy chief of mission, and Hubert Saint-Macary as a Paris morgue director.
The first installment in the Jason Bourne film series, it was followed by The Bourne Supremacy (2004), The Bourne Ultimatum (2007), The Bourne Legacy (2012), and Jason Bourne (2016).