Orphaned brothers Sam and Nathan "Nate" Drake are caught trying to steal a map made after the Magellan expedition from a Boston museum. Before the orphanage can expel Sam, he sneaks out to be on his own, but promises Nate that he will return, leaving him a ring belonging to their ancestor Sir Francis Drake (although Sir Francis Drake actually had no children).

Fifteen years later, Nate works as a bartender in New York City and pickpockets wealthy patrons. Victor "Sully" Sullivan, a fortune hunter who worked with Sam tracking treasure hidden by the Magellan crew, explains to Nate that Sam vanished after helping him steal Juan Sebastian Elcano's diary. Nate, having stopped receiving postcards from Sam, agrees to help Sully find him. Sully and Nate go to an auction to steal a golden cross linked to the Magellan crew, where they meet Santiago Moncada, the last descendant of the Moncada family, who had financed Magellan‘s expedition, and Jo Braddock, leader of Moncada's mercenaries. Nate is ambushed by Braddock's men, and the ensuing fight creates a distraction for Sully to steal the cross.

The duo travel to Barcelona, where the treasure is supposedly hidden, and rendezvous with Sully's contact Chloe Frazer, who has another cross. Nate, Chloe, and Sully follow clues in Elcano's diary to Santa Maria del Pi, finding a secret crypt behind the altar. Nate and Chloe enter, finding a trap door, but as they open it, the crypt floods with water. Sully helps them escape after subduing an ambush by Braddock. Using the two crosses to unlock a secret passage, Nate and Chloe find a map indicating the treasure is in the Philippines. Chloe betrays Nate and leaves to take the map to Moncada, hinting Sully is keeping a secret about Sam.

Sully recovers Nate and reveals that Sam was shot and apparently killed by Braddock three years prior and that he left him for dead, straining their partnership. Moncada, Chloe, and Braddock's team depart in a cargo plane to find the treasure, where Braddock kills Moncada, gaining control of the operation. After Nate and Sully board the plane, Nate confronts Braddock. A battle ensues; Sully parachutes out with the map, while Nate and Chloe are ejected from the plane landing in the Philippines, where they realize the map does not pinpoint the treasure. Nate discovers the treasure's true location through hints left by Sam's postcards, but leaves fake coordinates for Chloe after correctly doubting her loyalties. He discovers the Magellan ships and reunites with Sully. Braddock follows them, forcing Nate and Sully to hide as her crew airlifts the ships.

In their escape, Sully commandeers one of the helicopters, and Braddock orders another helicopter to approach for a boarding action. Nate defends himself from her mercenaries and shoots down the other helicopter with one of the ship's cannons. When Braddock corners Nate, Sully throws a bag of collected treasure at her, casting her into the sea, where she is killed when the ship falls on her. As Filipino naval units arrive, Nate and Sully get away with a few pieces of pickpocketed treasure while an arriving Chloe is left empty handed. Meanwhile, an imprisoned Sam, revealed to be alive & had somehow survived being shot by Braddock, writes another postcard to Nate asking him to watch his back.