Gru, a former villain turned Anti-Villain League (AVL) agent, faces a new challenge when he is fired from the AVL. He and his wife Lucy fail to capture Balthazar Bratt, a former child star turned supervillain obsessed with the character he played in the '80s. Bratt manages to steal a very expensive diamond, which leads to Gru and Lucy's termination given their failure to apprehend him. Dejected, Gru struggles with the idea of going back to his villainous ways despite the temptation and urging from his Minions. In the midst of this crisis, Gru discovers he has a twin brother.

Gru meets his long-lost twin, Dru, who is blond, cheerful, and successful, with an enormous, lavish mansion and a private army of minions. Dru reveals their father was a legendary villain, and he wishes to continue the family legacy. He admires Gru's past as a villain and seeks his mentorship to learn the ways of villainy. Gru is initially reluctant but is persuaded by the prospect of using this opportunity to get back at Bratt for his earlier defeat and the loss of his job. Meanwhile, Lucy is finding her feet as a new mom to Gru's adopted daughters, Margo, Edith, and Agnes, and Gru's Minions, longing for a return to villainy, find themselves on their own adventure.