- Guardians of the Galaxy (first appearance; assembled)
- Fantastic Four
- Mr. Fantastic / Reed Richards
- Invisible Woman / Susan Storm
- Human Torch / Johnny Storm
- Thing / Ben Grimm
- Shi'ar Empire
- D'Ken Neramani (first appearance)
- Lilandra Neramani (first appearance)
- Kree Empire
- Ronan the Accuser (hologram only)
- Korath the Pursuer (first appearance)
- Norman Osborn
- Super-Slayer (single appearance; destruction)
- U.S.A. President
- Clayton Quill (picture only)
- Harry Osborn
While on their way for another day in Baxter Institute, Nathan Quill and Zoe Gillan are watching news about the Fantastic Four with the U.S.A. President welcoming D'Ken Neramani, the emperor of the Shi'ar Empire, and his daughter Princess Lilandra to Earth, as they overhear of the Shi'ar Imperials seeking to start a new peace treating with the humans. Only Norman Osborn (who is at the time answering for allegations for his previous attempt against Baxter Institute) does not believe their claims. Later on, Nathan and Zoe are surprisingly visited by Lilandra, who is interested in overseeing the students' scientific projects and share some knowledge from the Shi'ar. Just then, they witness the arrival of Kree official Korath the Pursuer, who had arrived there under Ronan the Accuser's demands to kill Lilandra and frame the humans.
Suiting up for battle, Star-Lord and Gamora stand in Lilandra's defense and duel against Korath. The young couple are soon joined by Drax the Destroyer, an altered bulldog and Nathan's pet companion, and Rocket Raccoon, and they force Korath to retreat. Lilandra also tells the group that the Kree Empire has been in war with countless alien races, including the Shi'ar, the Skrulls, and the people of Xandar, whom Ronan has become obsessed with committing genocide of following the deaths of his father and grandfather during the past conflicts. As the Fantastic Four are reported to be aiding D'Ken in defending the Shi'ar Empire from Ronan's fleet, Nathan, Zoe and their companions stay to protect Lilandra by any means. Rocket also reveals that Korath is also after some Kree relics stored in Oscorp and plans to deliver them to the Kree scientists so they can prepare to launch their full attack on Earth. Invading one of Oscorp's laboratories, they find some of the scientists having been infected by a virus coming from the relics, and fight them off. They are also joined by Groot, a sentient humanoid tree from Asgard, and Star-Lord's old mentor Yondu Udonta, and defeat the mutated scientists, who are restored to normal once the stored relic is destroyed by Groot. One of the healed scientists barely recovers and reveals that Osborn has ordered the creation of the Super-Slayer, which is meant to be Oscorp's "ultimate defense unit", using the Kree relics as batteries. Having discovered how unstable the relics are, Star-Lord and Gamora are certain, however, that the robot will be too dangerous to be among both humans and aliens if activated.
The group moves on to the central Oscorp facility, where the Super-Slayer is being prepared. They are ambushed by Korath and a pack of Sakaarian pirates, but fight them off while Groot teleports Lilandra safely to Asgard. Meanwhile, the Fantastic Four are with the Shi'ar Royal Guard holding their own against Ronan's Kree armada before being informed of Lilandra's safety and are joined in the battle by Groot. Seeking to take advantage of the Fantastic Four's absence from Earth, Osborn intends to announce the Super-Slayer to the crowd as their "new protector", despite the scientists warning him that the prototype is not ready. Just then, the citizens witness Star-Lord, Gamora, Rocket, Yondu, and Drax fighting Korath's crew and cheer on them, ignoring Osborn's announcement in the progress. Angered, Osborn (against others' warnings) orders his employees to connect one of the Kree relics to the Super-Slayer and activate it. The Super-Slayer awakens, but instead of obeying Osborn's orders due to the relics being too unstable, it begins wreaking havoc in the city, alerting the heroes and drawing Korath's attention. Osborn tries to call the Super-Slayer off, but is ignored as he watches in horror his son Harry at the Slayer's aim.
Star-Lord and his team arrive in time to save and escort Harry to safety and fight the robot off. Korath aims to retrieve the relic, but is called off by Ronan, who decides to let the Super-Slayer use the relic so that it can destroy the earthlings and the Shi'ar Imperials for the Kree, and flees from Earth with the Sakaarians. Groot arrives with the Fantastic Four to help fight off the Super-Slayer and, right after Rocket and Drax make it inside the robot's body and destroy the Kree relic in it, the heroes combine their efforts to destroy the robot and save the city. As Osborn faces accusations for the incident and is demanded to shut down his Super-Slayer project for the safety of all, Star-Lord and his team are hailed by the crowd, the Fantastic Four and the Shi'ar Imperials as heroes (addressed by media as the "Guardians of the Galaxy") while D'Ken announces that the Shi'ar forces will be allied with Earth's military forces should the Kree be a threat to them again. Star-Lord and the Guardians set out for their first adventure in space together.
- Scott Menville as Star-Lord / Nathan Quill
- Laura Bailey as Gamora / Zoe Gillan
- Troy Baker as Rocket Raccoon / Sebastian Allan
- Travis Willingham as Groot
- William Salyers as Yondu Udonta
- Alan Tudyk as Mr. Fantastic / Reed Richards
- Catherine Taber as Invisible Woman / Susan Storm
- Max Mittelman as Human Torch / Johnny Storm
- Dave Boat as Thing / Ben Grimm
- Kari Wahlgren as Princess Lilandra
- David Sobolov as Emperor D'Ken Neramani, Super-Slayer A.I.
- Jonathan Adams as Ronan the Accuser
- Jeffrey Combs as Norman Osborn
- Dave Fennoy as Korath the Pursuer
- Sam Riegel as Harry Osborn
- The title is a reference to a trailer soundtrack produced by Audiomachine.
- "Because We Can" by Bon Jovi