A mysterious aircraft, using a giant magnet, steals a highly potent mutagen known as PX-41 from a secret laboratory in the Arctic Circle. Director Silas Ramsbottom of the Anti-Villain League (AVL) sends one of his agents, Lucy Wilde, to recruit Gru, a former supervillain. Forcibly brought to the AVL headquarters, Gru is asked by Silas to help them track down the culprit and recover the mutagen. However, Gru refuses, claiming that he is a legitimate father. Longing to resume his criminal life, Dr. Nefario, Gru's friend and assistant, leaves him for new employment. This causes a reluctant Gru to investigate the theft and work with Lucy. The pair are stationed at the Paradise Shopping Mall, with a cupcake store as their front.
Gru immediately suspects Mexican restaurant owner, Eduardo Pérez, of being "El Macho", a supervillain who supposedly died by riding a shark into an active volcano with 250 pounds of TNT strapped to his chest. Gru and Lucy break into Eduardo's restaurant at night but find no proof. Meanwhile, Gru's youngest daughter Agnes, who dreams of having a mother one day, believes that Gru will fall in love with Lucy. Gru denies it, saying his relationship with Lucy is only professional.
Despite holding Eduardo as his prime suspect, Gru agrees to pursue others, including wig merchant Floyd Eaglesan, where he discovers traces of PX-41. After witnessing Eduardo's son, Antonio, woo Margo and invite everyone to his Cinco de Mayo party, Gru renews his focus on Eduardo. After a neighbor sets Gru up on a bad blind date, Lucy helps him escape, and the two begin to bond. The next day, the AVL arrests Floyd after finding an almost-empty jar of the mutagen in his shop. Silas closes the investigation and reassigns Lucy to Australia.
At the Cinco de Mayo party, Gru follows Eduardo and discovers that he is indeed El Macho. Having faked his death, El Macho has hired Dr. Nefario, and has been abducting Gru's Minions, using the stolen PX-41 serum to transform them into monsters. El Macho plans to launch rockets full of the evil Minions into major cities to dominate the world. He offers Gru the chance to team up with him, but he walks away with the girls. El Macho sends one of his evil Minions after him.
Lucy later arrives at the party right after Gru leaves, but El Macho captures her. Dr. Nefario lets Gru know what happened, and he returns to El Macho's fortress with two of his own disguised Minions, but the evil Minions remove their disguise and attack them. Meanwhile, the first evil Minion attacks the girls at Gru's house, until Dr. Nefario, who has returned to Gru's side, arrives with an antidote. Dr. Nefario puts the antidote in Gru's jelly reserves, and he and the girls hurry to Gru's aid. Gru, Dr. Nefario, and the girls use jelly guns to restore all the Minions to their normal state. El Macho applies the mutagen himself to attack Gru, but he and Dr. Nefario defeat him.
Gru starts to rescue Lucy from El Macho's TNT-loaded shark rocket, but his pet chicken Pollito lauches it, sending them towards a volcano. Lucy accepts Gru's invitation for a date, and they jump into the ocean before the rocket explodes in the volcano. Gru joins the AVL, and he and Lucy eventually get married, giving Margo, Edith, and Agnes a mother.
Steve Carell as Gru
Kristen Wiig as Lucy Wilde,
Benjamin Bratt as Eduardo Pérez / El Macho,
Miranda Cosgrove as Margo,
Dana Gaier as Edith,
Elsie Fisher as Agnes,
Russell Brand as Dr. Nefario,
Steve Coogan as Silas Ramsbottom,
Ken Jeong as Floyd Eaglesan
Moisés Arias as Antonio Pérez,
Nasim Pedrad as Jillian,
Kristen Schaal as Shannon,
Pierre Coffin as Kevin, Stuart, Bob, and additional Minions.
Chris Renaud as additional Minions
Renaud also voices an Italian waiter
Vanessa Bayer as a flight attendant
Nickolai Stoilov as the Arctic Lab guards