The G.I. Joe are sent to find Cobra Commander, but one of the members, the heart of the G.I. Joe, is killed in action.
- Conrad S. Hauser/Duke (Chris Evans) - The main protagonist and a biological clone created by COBRA scintest, Dr. Minbender after extracting his blood and hair samples from the air raid commanded by Zortan, hoping that Duke that accomplish future goals for Cobra, after rememberig the bombing, he escapes and. In the end, he and Roadbluck get promoted and he and Scarlett shared a romantic kiss. Chris Evans takes Channing Tatum's place in this third sequel as Duke's clone
- Marvin F. Hinton/Roadblock (Dwayne Johnson) - leader of the G.I. Joes, he shows great trust towards Dusty, and accidentally got him killed by Cobra, in the end, gets promoted!
- Ronald W. Tadur/Dusty/X (Shia LaBeouf) - trusted by Roadblock, he has a hawaiian girlfriend named Darla, he wrote a letter to her, saying that it would be his final time as a Joe, he is killed by Cobra, who threatened to burn the letter he kept in his pocket to give to The President, as he is forced to cooperate, revealed near the end, that he didn't die, as Block faked his death, with a fake blood weapon to keep to fake his death, to move slightly from "Cobra Commander", and he infiltrated Cobra, as a new member, "X", to spy on Serpentor with the weapon Jaye used to scan Zartan in "G.I. Joe: Retaliation", to reveal that Serpentor was a clone, in the end, goes back to his girl in Hawaii!
- Dashiell R. Faireborn/Flint (D.J. Cotrona) - angry at Snake Eyes and Scarlett for not showing up earlier, until he is lectured and punched by Lady Jaye, Jaye told him others did what did, because they care, and Flint questions, "Like you do", as they start to have a tender moment by hugging, in front of Snake Eyes and Scarlett, in the end, is seen knocking Tomax out cold!
- Alison R. Hart-Burnett/Lady Jaye (Adrienne Palicki) - consoles Flint, as they had a tender moment hugging, in front of Snake Eyes and Scarlett, in the end, She is seen looking in Serpentor's file!
- Snake Eyes (Ray Park) - reveals his face to Scarlett, revealing a scar over his eye, and lip, which was from his first fight against Stormshadow, in the end, He leaves for the Indies!
- Shana M. O'Hara/Scarlett (Rachel Nichols) - Duke's love interest of the film and Ripcord's ex-girlfriend. realizes how Snake Eyes was when his faced was scarred, in the end, she reveal to duke that the reason that she and Ripcord broke up is that he cheated on her.
- Wayne R. Sneeden/Beach Head (Ryan Reynolds) - a headstrong member of the G.I. Joe, but is loyal to his team, in the end,
- David L. Katzenbogen/Bazooka (Ben Stiller) - dosen't seem to like Alpine very much, because he thinks he's white, in the end, batters Xamot!
- Albert M. Pine/Alpine (Don Cheadle) - dosen't seem to get along well with Bazooka, because he's white, in the end, gets over his racial attitude!
- Ettienne R. LaFitte/Gung-Ho (Jesse Ventura) - appears as a badass, plain and simple, he finds throwing down with some COBRA bad guys in a street-style fight would be immensely enjoyable, in the end,
- Sgt. Slaughter (Himself) - trains the Joes and is very close with Beach Head, in the end,
- Hector X. Delgado/Shipwreck (Will Wheaton) - had history with Alpine and Snake Eyes, revealing that they were trained together, in the end, returns to the Medditeranian!
- General Lawrence J. Flagg (Bruce Greenwood) - sent the Joes on the mission to find Cobra, in the end,
- Junkyard (Chuck Norris) - has a pet dog, Mutt, in the end,
- William S. Hardy/Wild Bill (Owen Wilson) - aids the Joes in a manhunt for Cobra, in the end,
- Carl W. Greer/Doc (Terrence Howard) - doctor of the Joes, in the end,
- Serpentor (Ron Perlman) - the main antagonist, meets with "Cobra Commander", his personality include genius, ruthfulness, daring, and shrewdness, his goal is to overthrow Cobra Commander, as he would be a ultimate leader, in the end, he realized that the Cobra he killed was Fred VII as he recognized that Cobra would never die breathing in his mask, and learned that Cobra had created him with the genius of Napoleon I of France, the ruthfulness of Julius Caesar, the daring of Hannibal and the shrewdness of Attila the Hun,
- Rex Lewis/Cobra Commander (Luke Bracey/Robert Baker) - had Fred cover him as Cobra Commander, he still learned nanomite technology from Dr. Mindbender,
- Anastasia Cisarovna/Baroness II (Yuliya Snigir) - Cobra's second-in-command and new Baroness, after his sister, Ana, cares more for Duke and less about Destro, unlike her, Baroness II cares about Destro. In the end,
- James McCullen/Destro (Christopher Eccleston) - Cobra's weapon supplier, though distrusts Cobra Commander because he betrayed him twice by turning him into Destro and overthrowing him "G.I. Joe: Rise of Cobra" and leaving him in prison in "G.I. Joe: Retaliation", shows tenderness towards the new Baroness than Ana, after she heartbroken him for Duke, in the end,
- Tomax and Xamot (James and Oliver Phelps) - twin brothers and legitimate businessmen to be the respectable "face" of their operations, in the end,
- Sebastion Bludd/Major Bludd (Sam Worthington) - Cobra's hired assassin, as he kills Dusty to cooperate where the Joes are, he would disobey Cobra, in the end,
- B.A.T. (short for Battle Android Troopers)
- Fred VII (Robert Torti) - was seen near the end disguised as Cobra Commander, playing nice with Serpentor, and ended up betrayed and killed, as he couldn't breath through the mask
- Zarana (Moon Bloodgood) - the new leader of the Dreadnoks, whose main objective is to kill the G.I. Joes for the deaths of her brother, Zartan and her twin, Zandar in "G.I. Joe: Retaliation", she whistles "For He's a Jolly Good Fellow" like her brother, and smokes a cigarette, in the end,
- Golobulus (Patrick Stewart) - ruler of Cobra-La, was seen at the end,
- Pythona (Cate Blanchet) - Golobulus' assassin' seen at the end,
- Darla (Vanessa Hudgens) - Ronald's hawaiian girlfriend, in the end,