Once upon a time, Gru (voice of Steve Carrell) dreamt of stealing the moon - these days he's a single adoptive father of three precocious girls. Having given up his spectacular life of crime, Gru now aims to produce a delicious new line of jams with the help of his tiny minions and his old pal Dr. Nefario (voice of Russell Brand). Feeling unfulfilled in his newfound role as a jam-maker, Dr. Nefario announces that he will be departing for more evil pastures just as Gru is approached by the Anti-Villain League to capture the mysterious culprit who just stole a top-secret research lab filled with PX 41 - an experimental serum with the strength to transform harmless animals into ravenous monsters. The AVL is certain that the culprit is covering his tracks by posing as a small business owner at a local mall, and assign their best agent Lucy (Kristen Wiig) to aid Gru in identifying him. Meanwhile, Gru's oldest adopted daughter Margo (voice of Miranda Cosgrove) falls for rebellious teen Antonio (voice of Moises Arias), the leather-jacketed son of boisterous Mexican cantina owner Eduardo (voice of Benjamin Bratt). Now, with Gru's paternal instincts kicking into full gear just as a dastardly plot begins to unfold, the devoted new father must divide his time between saving the world from an enigmatic super-villain, and saving Miranda from total heartbreak. All the while, a suitable mother for the girls may have been standing right under Gru's pointy nose the entire time.
While Gru, the ex-supervillain is adjusting to family life and an attempted honest living in the jam business, a secret Arctic laboratory is stolen. The Anti-Villain League decides it needs an insider's help and recruits Gru in the investigation. Together with the eccentric AVL agent, Lucy Wilde, Gru concludes that his prime suspect is the presumed dead supervillain, El Macho, whose his teenage son is also making the moves on his eldest daughter, Margo. Seemingly blinded by his overprotectiveness of his children and his growing mutual attraction to Lucy, Gru seems on the wrong track even as his minions are being quietly kidnapped en masse for some malevolent purpose.