Following the events of The Rise of Cobra, Duke (Channing Tatum) has become the leader of the G.I. Joe unit, which is framed for stealing nuclear warheads from Pakistan by Zartan (Arnold Vosloo), who is impersonating the President of the United States (Jonathan Pryce). The unit is subsequently eliminated in a military air strike with Duke as one of the casualties. The only survivors are Roadblock (Dwayne Johnson), Flint (D.J. Cotrona), and Lady Jaye (Adrianne Palicki).
Meanwhile, Storm Shadow (Byung-hun Lee) and Firefly (Ray Stevenson) rescue Cobra Commander (Luke Bracey) from an underground maximum-security prison in Germany, leaving Destro behind. 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 (Élodie 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 elite special forces 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. Lady Jaye steals a sample of the President’s DNA and confirms that he is Zartan. They escape after a confrontation with Firefly and Zandar (Matt Gerald), the head of the U.S. Secret Service’s Presidential Detail and a member of Cobra.
Snake Eyes and Jinx locate and capture Storm Shadow after a battle with ninjas and take him to Japan, where Storm Shadow reveals that Zartan murdered the Hard Master, and that he joined Cobra to avenge his uncle. Storm Shadow then accompanies Snake Eyes and Jinx as they join the Joes’ efforts to stop Cobra.