The G.I. Joes are framed for stealing nuclear warheads from Pakistan by Zartan (Arnold Vosloo) who is impersonating the President of the United States (Jonathan Pryce). The entire team is eliminated in a military strike with Duke (Channing Tatum) as one of the casualties. Roadblock (Dwayne Johnson), Flint (D.J. Cotrona) and Lady Jaye (Adrianne Palicki) are the only survivors.
Meanwhile, Storm Shadow (Lee Byung-hun) and ex-Joe Firefly (Ray Stevenson) rescue Cobra Commander (Luke Bracey) from a penitentiary in Germany. Storm Shadow is injured during the escape and retreats to a temple in the Himalayas to recover. Upon learning that he is alive, the Blind Master (RZA), leader of the Arashikage Clan, sends Snake Eyes (Ray Park) and his apprentice Jinx (Elodie Yung), Storm Shadow's cousin, to capture Storm Shadow so he can answer for the murder of his uncle, the Hard Master.
Roadblock, Flint, and Lady Jaye return to the United States where they set up a base of operations in a rundown gym. After Zartan announces that Cobra will replace the Joes as America's main protective unit, Lady Jaye deduces that someone is impersonating the President and Roadblock leads them to General Joseph Colton (Bruce Willis) who provides them with weapons and helps them infiltrate a fundraising event that the President will be attending, where Lady Jaye steals a sample of his DNA and confirms that he is Zartan. They escape after a brief confrontation with Firefly and Zandar (Matt Gerald), the head of the Presidential Detail and a member of Cobra.
Snake Eyes and Jinx locate and capture Storm Shadow after a battle with ninjas and take him back to Japan where Storm Shadow reveals that Zartan murdered the Hard Master and he joined Cobra to avenge his uncle, embittered that the clan didn't believe in his innocence. Storm Shadow then accompanies Snake Eyes and Jinx as they join the Joes' efforts to stop Cobra.
Zartan invites the world leaders to a summit, where he blackmails them into disabling their nuclear arsenals, and reveals that he has created Project Zeus: Seven orbital kinetic bombardment weapons of mass destruction at his command. He destroys central London to prove his superiority and threatens to destroy other capitals if the countries don't submit to Cobra. However, Storm Shadow betrays Cobra Commander and kills Zartan, revealing Cobra's deception to the world leaders. While Snake Eyes, Jinx and Flint fight Cobra's soldiers, Cobra Commander activates the remaining six weapons and instructs Firefly to protect the launch device. Firefly is killed in combat with Roadblock, who deactivates and destroys the orbital weapons. Meanwhile, Colton and Lady Jaye rescue the President.
Cobra Commander escapes during the battle and Storm Shadow disappears after avenging his uncle. The President initiates a global massive manhunt on Cobra Commander, who is now considered to be a terrorist, and reinstates the Joes with Roadblock leading Flint, Lady Jaye, Snake Eyes and Jinx, and recruiting more soldiers to join them. Roadblock swears to avenge Duke by capturing Cobra Commander.
- Bruce Willis as General Joseph Colton
- Dwayne Johnson as Roadblock / Marvin F. Hinton
- D.J. Cotrona as Flint
- Lee Byung-hun as Storm Shadow
- Adrianne Palicki as Lady Jaye / Jaye Burnett
- Ray Park as Snake Eyes
- Jonathan Pryce as the President of the United States
- Ray Stevenson as Firefly
- Channing Tatum as Duke / Conrad S. Hauser
- Arnold Vosloo as Zartan
- RZA as Blind Master
- Elodie Yung as Jinx
- Joseph Mazzello as Mouse
- Walton Goggins as Warden Nigel James
- Robert Catrini as the Israeli President
- Luke Bracey as Cobra Commander
- Robert Baker as the voice of Cobra Commander