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), headed by Silas Ramsbottom, tries to recruit former supervillain Gru to track down those responsible and recover the mutagen, but Gru refuses, claiming that he is now a legitimate businessman as well as the devoted father of Margo, Edith, and Agnes. Dr. Nefario, Gru’s friend and assistant, “misses being evil” and leaves Gru for new employment. Gru reluctantly agrees to investigate the theft, and is partnered with undercover AVL agent Lucy Wilde.
The pair begin their search in the Paradise Shopping Mall, disguised as bakery employees. Gru immediately comes to suspect Mexican restaurant owner, Eduardo Pérez, of being the infamous “El Macho”, a legendary supervillain who supposedly died after riding a shark into the center of an active volcano with dynamite strapped to his chest. Gru and Lucy break into Eduardo’s restaurant at night, but find no proof. Meanwhile, Agnes, confiding in her dream to one day have a mother, believes that Gru will fall in love with Lucy, though Gru states that his relationship with Lucy is professional.
Although holding Eduardo as his chief suspect, Gru agrees to pursue others, including wig merchant Floyd Eagle-san. After witnessing Eduardo’s two-timing son, Antonio, woo Margo and invite everyone to his Cinco de Mayo party, Gru renews his focus on Eduardo. He is then set up on a dinner date with a vacuous woman named Shannon. The date goes horribly, and Shannon is about to humiliate Gru after she spots his wig, until Lucy, visiting the restaurant herself, saves Gru and renders Shannon unconscious with a tranquilizer gun. While they take Shannon home, Gru and Lucy spend personal time together, and Gru falls in love with her, proving Agnes right.