Duke (Conrad Hauser) - The lead soldier. He is part of General Hawk's team. Contrary to his original back-story, he is as a newcomer to the G.I.Joe team; all Joe rookies are already established, well-trained soldiers. Also in the 2009 film, Duke identifies himself as a Captain while conspicuously wearing the rank of Major (Army) or Lt. Commander (Navy). There is no reference in the film to him ever performing duties as a First Sergeant, the rank of the original Duke character in the GI Joe animated series.
General Hawk (Gen. Clayton Abernathy) - The team leader. He is the commander of the G.I. Joe Team.
Ripcord (Wallace Weems) - Duke's friend, a pilot with a loving interest on Scarlett. Also a newcomer to G.I. Joe, and one of the film's major characters; the traditional Rip Cord, however, is caucasian, a G.I. Joe veteran, and more of a background character. Before joining G.I. Joe, they were mid-level ground troops in the U.S. Army. Unlike Duke, Ripcord loves flying planes and wanted to join the U.S. Air Force before becoming a Joe.
Scarlett (Shana M. O'Hara) - She graduated college at age twelve and became the team's intelligence expert. Having left school so early, she does not understand men's attraction to her. Whether she and Snake Eyes are in a relationship with each other is unknown.
Snake Eyes - A mysterious ninja commando who took a vow of silence, a departure from the character's traditional difficulty in speaking due to grievous vocal wounds suffered in a helicopter crash.
Heavy Duty (Hershel Dalton) - An ordnance expert and field commander of the team.
Breaker (Abel Shaz) - He is the team's communications specialist and hacker.
Cover Girl (Courtney A. Kreiger) - Hawk's aide-de-camp.
Sgt. Stone - A special operations instructor who trains the G.I. Joe team in combat tactics, marksmanship, survival techniques, and other commando skills.
The Doctor / Cobra Commander (Alexander "Rex" Lewis) - The leader of Cobra and The Baroness's brother, a former mild-mannered U.S. Soldier who was thought to be killed during an operation - instead, he became the insane disfigured MARS head scientist.
Destro (Laird James McCullen XXIV) - A weapons designer and CEO of the Military Armament Research Syndicate (MARS) and the main villain in the early part of the film.
The Baroness (Anastasia Lewis "Ana" DeCobray) - A spy and sister of Cobra Commander who is also both Duke's former fiancee and his and Destro's love interest.
Storm Shadow (Thomas "Tommy" Arashikage) - Snake-Eyes' rival, both were close members of the Arashikage ninja clan. Storm Shadow serves as McCullen's assassin and as both bodyguard and mentor to the Baroness.
Zartan - An expert in make-up and disguises serving Destro.
Doctor Mindbender also appears in a flashback, as it is revealed that he passed the secrets of nano-technology on to Cobra Commander.