A mysterious vehicle using a huge magnet steals a highly potent mutagen known as PX-41 from a secret laboratory in the Arctic Circle. The Anti-Villain League (AVL) tries to recruit former supervillain, Gru, to find who stole the mutagen, but Gru refuses, claiming he is a legitimate businessman now as well as the father of Margo, Edith, and Agnes. Dr. Nefario, Gru’s friend and assistant, has decided to leave him for new employment, claiming he “missed being evil.” Gru reluctantly partners with undercover AVL agent Lucy Wilde, and they search a local mall, the Paradise Shopping Mall, disguised as bakery employees. Mexican restaurant owner, Eduardo Perez, is suspected by Gru of being “El Macho”, a legendary supervillain who supposedly died after skysurfing a shark into the center of an active volcano. Gru and Lucy break into Eduardo’s restaurant at night, but find nothing to prove that he is the culprit. Meanwhile, Agnes expresses her wish to have a mother someday and believes that Gru will fall in love with Lucy, but despite her excitement, Gru tells her that his relationship with Lucy is strictly professional.
Gru and Lucy investigate wig merchant Floyd Eagle-san, but Gru is still suspicious of Eduardo after seeing Eduardo’s two-timing son, Antonio, going out with Margo, and inviting everyone to his Cinco de Mayo party. Afterwards, Gru is set up on a horrible dinner date with a rude woman named Shannon, who notices Gru’s wig and threatens to humiliate him, but Lucy rescues him by shooting Shannon with a tranquilizer dart. They take Shannon home, and afterwards, Gru realizes that Agnes was right, as he has fallen in love with Lucy.
The next day, the AVL arrests Floyd Eagle-san due to an almost-empty mutagen jar being found in his shop, and the investigation is closed. Lucy is reassigned to Australia, but before leaving, gives Gru her lipstick Taser to remind him of her. This leaves Gru heartbroken because he loves her, but he cannot find the courage to ask her out on a date. Instead, he brings the girls to the Cinco de Mayo party and finds proof that Eduardo is El Macho. Gru discovers that he and his partner, Nefario, have captured and mutated a large number of Gru’s Minions using the PX-41, turning them into insane, savage purple-furred monsters, with El Macho planning to send rockets full of mutated Minions to major cities and achieve world domination. El Macho proposes that he and Gru team up, but Gru avoids answering and leaves.
On her flight to Australia, Lucy realizes she has also fallen in love with Gru and jumps out of the plane, but after hang gliding down to the party, she is captured by El Macho after Pollito, his pet chicken, retrieves her AVL ID badge from her purse. Fortunately, Nefario informs Gru, and to rescue Lucy, Gru visits Macho along with two Minions covered in purple paint, pretending he was captured by them. Fighting alongside his daughters and Nefario, Gru and his team spray all of the mutated Minions with jelly containing a powerful antidote that Dr. Nefario made, whereupon they revert to their friendly yellow state. Macho then takes the mutagen himself, but Gru and Dr. Nefario overcome him using Lucy’s lipstick Taser and a fart gun.
Gru sees Lucy strapped to a TNT-loaded shark rocket and starts to untie her, but Pollito launches the rocket, sending the rocket flying towards the same volcano where Macho previously faked his death. Lucy accepts Gru’s invitation for a date, and the pair dive into the ocean seconds before the rocket enters the volcano.
After 147 dates, Gru and Lucy are married and Margo, Edith, and Agnes finally have a mother. The Minions close with a rendition of “I Swear” and “Y.M.C.A” as the whole family celebrates.