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The film revolves around an special FBI organization of trained secret agent animals, equipped with advanced tools including an advanced earpiece that allows the mammalian members to talk to humans. In addition to a team of cockroaches, the primary field team consists of guinea pigs Darwin (Sam Rockwell) (team leader), Juarez (Penelope Cruz) (martial arts), Blaster (Tracy Morgan) (weapons/transportation), star-nosed mole Speckles (Nicolas Cage) (cyber intelligence), and fly Mooch (reconnaissance) (Dee Bradley Baker). Hoping to impress his superiors on the eve of a budgetary review, the unit's leader, Ben (Zach Galifinakis), orders an unauthorized infiltration of the residence of home electronics and appliances magnate, Leonard Saber - Owner of Saberling Technology, who has been under FBI investigation for years. The team is able to successfully retrieve considerable sensitive information about a sinister scheme that is set to occur in 29 hours. However, when Ben's superior arrives for his evaluation, his astonishment at the team's capabilities and technology is overcome by his indignation at Ben's unauthorized mission and the fact that the downloaded intelligence appears to be useless information about Saber's coffee makers. As a result, the government agent orders the unit shut down, the equipment seized and the animals to be used as experimental subjects to be killed as security risks. With the help of their human compatriots, Darwin, Juarez, Blaster, Mooch, and Speckles escape with hopes of stopping Saber's scheme, but find themselves in a pet carrying case bound for a pet shop.
Now trapped in the store's pet rodent display case, G-Force meets Hurley (Jon Favreau), a gluttonous guinea pig, Bucky (Steve Buscemi) an irascible hamster and three sycophantic mice. Although Blaster and Juarez manage to get themselves sold to a family with plans to return to extract their comrades, Speckles' own attempt to escape by playing dead ends disastrously when he is thrown into and apparently crushed in a garbage truck. Meanwhile, Mooch manages to return to Ben to tell him where his mammalian agents are, but Darwin escapes (with Hurley, who is convinced that Darwin is his brother, tagging along) before he can arrive to collect them.
While Blaster and Juarez escape their new owners to return to Ben, he and his partner discover that the discredited intel has a destructive computer function that apparently hid the scheme. At this time, Darwin and Hurley make their own way to their superior. On route, Darwin sees a Saber coffeemaker and decides to investigate it, but his examination of the machine makes it come alive as a dangerous fighting robot that he and Hurley are barely able to defeat. Now with his suspicions vindicated, Darwin and Hurley transport the wreckage to Ben. However, upon arrival, Ben has lost all confidence in his team and confesses the shattering information that they are not special genetically enhanced animals as previously told, but ordinary ones Ben took in and trained for the team. However, Hurley lifts them from their despair by reminding the team of the astounding feats he has seen them do and the fact that they obviously made themselves extraordinary on their own.
Emboldened but with little time to stop the scheme, Ben provides the field team with the means to infiltrate the Saber residence and plant a virus in the computer mainframe. Unfortunately, FBI agents are ordered to capture the animals dead or alive, forcing the team to elude them with an extended pursuit thanks to a high speed vehicle especially designed for them. After that is accomplished and the team infiltrates Saber's mainframe, the plan is put into motion, and the resulting battle separates the group, only leaving Darwin to take the mainframe down. At the same time, Leonard Saber is shocked to discover that his appliances have become killing machines, expecting them to simply be able to effectively communicate with each other, while FBI takes advantage of this obvious pretext to finally openly move against the industrialist. When Darwin reaches the mainframe, he finds out that Speckles, whose home and family had been destroyed by humans, is the mastermind of the plan, whose masterstroke is to cause a massive planetwide bombardment of space junk pulled from orbit to make the planet surface uninhabitable. Speckles promptly amalgamates the various appliances in the vicinity into a giant walking being, which, combined with a localized bombardment of orbital debris, soon overpowers the police forces gathered at the mansion. Darwin manages to persuade Speckles that his new family is with the rest of the team and Ben, who had taken them all in. Speckles consents, and tries to shut it down, but realizes that it has gone too far. However, Darwin uses the computer virus on his PDA to take it down.
At the end of the film, the guinea pigs are personally commended by the FBI Director who also appoints them special agents of the FBI. Furthermore, G-Force is reinstated as a unit of the Bureau and expanded with Hurley, Bucky and the mice inducted as new recruits. Meanwhile, Saber makes the largest product recall in history, and Speckles is given the punitive duty of personally removing the malicious chips from all Saber products, which number in the tens of thousands.
