A mysterious aircraft swipes a dangerous mutagen named PX-41 from a lab located in the Arctic Circle. In response, Silas Ramsbottom, the head of the Anti-Villain League (AVL), sends agent Lucy Wilde to seek out Gru, a former supervillain. Despite being forcibly brought to AVL's headquarters, Gru resists their plea, insisting that he has enough on his plate as a father and as the owner of a jelly business. However, when his longtime colleague Dr. Nefario resigns to join another villain, Gru reluctantly decides to collaborate with the AVL, and he is paired with Lucy, using a cupcake store as their cover at a local shopping mall.
Gru quickly becomes suspicious of Eduardo Pérez, the owner of a Mexican restaurant, believing him to be the notorious supervillain "El Macho," who was thought to have perished in an explosive showdown with a shark and a volcano. Attempting to gather information, Gru and Lucy sneak into Eduardo's restaurant but come up empty-handed. Meanwhile, Gru's three adopted daughters—Margo, Edith, and Agnes—who long for a mother’s love, speculate about a potential romance between Gru and Lucy, an idea that Gru firmly dismisses as absurd.
While continuing to survey potential leads, Gru's focus remains on Eduardo, especially when he witnesses Eduardo's son Antonio charming Margo. Gru's neighbor, Jillian, inadvertently sets him up on a disastrous blind date with Shannon, but Lucy swoops in to rescue him from humiliation by tranquilizing Shannon. This incident strengthens the bond between Gru and Lucy, but their progress is interrupted when the AVL arrests Floyd, a wig merchant linked to the mutagen.
In a sudden turn, Ramsbottom shuts down the investigation and relocates Lucy to Australia, leaving Gru heartbroken. At a festive Cinco de Mayo party that Eduardo hosts, Gru confirms his suspicions when he discovers that Eduardo is indeed El Macho, who has survived and has enslaved Gru's Minions using the mutagen, transforming them into ferocious monsters poised for world domination.
El Macho extends an invite to Gru to join forces, but overcome with fear, Gru flees with his daughters. Meanwhile, Lucy, defying orders, rushes back to help them, only to be taken hostage by El Macho, a scene witnessed by Nefario. Gru, attempting to save her, feigns an abduction by two mutated Minions, only to be quickly unveiled by the real mutated Minions who launch an attack.
In a desperate race against time, Nefario devises an antidote to reverse the Minions' transformation. Nefario’s plan involves lacing Gru’s jelly with the antidote, and with the help of the girls and remaining Minions, they race to rescue Gru and restore the mutated Minions back to normal. A frantic showdown ensues as El Macho, threatened by Gru and Nefario's defiance, attempts to sacrifice Lucy to the volcano where he pretended to die, while also using the mutagen to battle Gru. Ultimately, Gru and Nefario emerge victorious.
As they try to rescue Lucy from a rocket rigged to launch her into the lava, the chaos leads to an unexpected twist. Lucy accepts Gru's romantic advance, resulting in a daring leap into the ocean just as the rocket is set to explode. Months later, after a whirlwind of one hundred forty-seven dates, love blossoms, culminating in a wedding where Gru and Lucy are united, and Lucy embraces the role of their girls' adoptive mother.
