Following the previous film, Gru is now an agent for the Anti-Villain League (AVL) and he and his wife Lucy are sent to foil the plans of Balthazar Bratt, a former child actor who portrayed a young supervillain in a popular television series “Evil Bratt” before it was cancelled as a result of his puberty, and his waning popularity led to him adopting his former persona to become an actual supervillain. Gru manages to stop Bratt from stealing a very expensive diamond, but is unable to capture him. As a result of constantly failing to capture Bratt, Gru and Lucy are fired from the AVL by its mean new director Valerie Da Vinci, following the retirement of Silas Ramsbottom.
Gru and Lucy reluctantly tell their children, Margo, Edith, and Agnes, of their termination, but assure them they will find new jobs someday. Already upset Dr. Nefario has frozen himself in carbonite, most of Gru’s Minions leave Gru when he refuses to return to villainy despite losing his job. Meanwhile, Lucy struggles to adapt in her new role as the children’s new mom. The next morning, the family is approached by Fritz, the butler of Gru’s long-lost twin brother Dru, who lives in a distant country named Freedonia and longs to meet him. Stunned at this revelation, Gru confronts his mother who reveals after divorcing Gru’s father, they agreed to keep one child each. The family decides to meet Dru and they are surprised at his immense wealth which Fritz attributes to their pig farm business. Meanwhile, the Minions are arrested after trespassing at a talent show set, while Bratt manages to steal the diamond once again, intending to use it to power a giant robot he constructed so he can destroy Hollywood as revenge for the cancellation of his show.
While Lucy takes the children around Freedonia, Dru reveals to Gru that their father was a legendary supervillain. As Dru was constantly dismissed by their father as a disgrace, he turned to his brother to teach him how to be a villain. When Gru refuses to revert back to his old ways, Dru brings him on a joyride around Freedonia in their father’s highly technological car where the siblings bond. Meanwhile, Lucy tries to balance her tasks as a mom which she exhibits after turning away a young boy named Nico who had fallen for Margo in the midst of a traditional cheese festival, all while Edith and Agnes stumble into a local tavern and learn from the bartender that a mythical unicorn lurks in the nearby forest. Gru, fatherly, attempts to convince Agnes that unicorns are imaginary, but decides not to on account of her anticipation. The next day, Agnes and Edith set out into the woods to track the mythical horse, only to find, unexpectedly, a one-horned goat whom Agnes dubs “Lucky” and takes to her heart. Meanwhile back in Los Angeles, the Minions exert control among the prisoners at their jail, but their longing for Gru motivates them to escape the prison.