A secret laboratory, in the middle of nowhere, has just been stolen by a mysterious vehicle using a giant magnet. The Anti-Villain League (AVL) become suspicious about this case as the lab contains a mutating chemical compound which could transform anything to become indestructible killing machines. The Anti-Villain League must find someone to stop the mutating chemical getting into the wrong hands.
Gru, formerly a super-villain, is having a birthday party for Agnes. He impersonates a Fairy Princess when the performer cannot attend. Much to Gru's chagrin, his annoying, overly-happy neighbor, Jillian, begins pushing Gru to take some of her single acquaintances on a date.
While he is outside, he and two of his minions are approached and kidnapped by Lucy Wilde, an Anti-Villain League agent, and taken to the agency's underwater headquarters, where they ask Gru to become a 'spy' in order to track down who has stolen the mutating chemical compound. Gru refuses, saying that he has other responsibilities now. Before he leaves, Lucy admits she was impressed by his work as a villain, stating that stealing the Moon was 'amazing', before giving him her name-card and urging him to contact her if he changed his mind.
Upon his return, Dr. Nefario confesses to Gru that he misses being 'evil' and has been offered employment elsewhere. He leaves Gru and his minions. This prompts Gru to contact Lucy and take the job. He works undercover in a mall as the owner of a cupcake shop. Lucy has been assigned as Gru's partner.
He suspects one of the owners of a restaurant, named Eduardo, in the mall to be an old super-villain called El Macho, who supposedly died after riding a shark with TNT into an active volcano. Lucy and Gru then decide to break into his restaurant and narrowly escape being caught, but find nothing to attach Eduardo to the crime.
Meanwhile, Margo begins to develop a crush on Eduardo's son, Antonio, to Gru's frustration. The whole family is invited to Eduardo's Cinco de Mayo party at his mansion.
Later that day, Jillian sets a reluctant Gru up on a date with her rude and superficial friend Shannon. During the date, Shannon discovers Gru's wig and threatens to humiliate him, but Lucy saves him by shooting her with a tranquilizing dart. They take Shannon home, and Gru tells Agnes that he thinks he is in love with Lucy.
When he arrives at the mall the next day, he sees the Anti-Villain League arrest the owner of a different store and tell Gru that the case is now closed and that Lucy will be getting posted in Australia for different work. Gru is not able to muster the courage to ask Lucy out and incinerates his telephone out of frustration.
The family then goes to Eduardo's party. Gru follows Eduardo and discovers that he was correct about Eduardo. Gru also discovers that Dr. Nefario has been working for Eduardo and that the duo has been capturing and mutating some of Gru's minions into the same purple-furred monsters as the serum creates.
Eduardo offers Gru a chance to work together and rule the world, but Gru makes his excuses and hastily leaves with the girls. Margo has also broken up with Antonio as he became interested in another girl.
Lucy, while in the plane bound for Australia, realized that she wants to be with Gru, and jumps out of the plane. She arrives at the party on a hang-glider, but Eduardo discovers who she is and captures her. Dr. Nefario calls Gru to tell him what has happened, and Gru takes two of his minions to get Lucy back.
Gru manages to restore the minions who were under El Macho's control using an antidote-containing jam which Dr. Nefario made and defeat El Macho. Lucy is seen tied to a rocket that is about to fly into the volcano where El Macho faked his death. Gru arrives on time to rescue her.
One hundred and forty-seven dates later, the two are married and the girls finally have a mother. The minions close with a rendition of Y.M.C.A while the family dances.
During the credits, 3 Minions are seen auditioning for the upcoming Minions movie and a black wall showing the credits keeps falling and blocking their way.