A supervillain named Felonius Gru has his pride injured when an unknown rival steals the Great Pyramid of Giza. With the assistance of his sidekick Dr. Nefario and his Minions, Gru resolves to one-up this mysterious rival by shrinking and stealing the Moon. Knowing this is extremely expensive, Gru seeks a loan from the Bank of Evil. Mr. Perkins, the bank president, is impressed by the plan, but he will approve the loan only if Gru can obtain the necessary shrink ray first. Upon learning that an up-and-coming villain known as Vector was responsible for the Pyramid theft, Gru openly displays a rivalry with him.
Gru and the Minions steal the shrink ray from a secret testing base in East Asia, but Vector intercepts them and steals it for himself. Gru attempts to break into Vector’s impregnable fortress to recover the shrink ray, only to be defeated by numerous defenses. As nothing seems to work, he notices three orphan girls Margo, Edith, and Agnes, who are able to easily enter the base as they are selling cookies. Gru disguises himself and adopts the girls, planning to use them to infiltrate Vector’s base. However, Gru has trouble nurturing them properly due to their stubbornness, their ballet classes and his own ineptitude as a parent.
Eventually, Gru and the girls arrive at Vector’s base, and Gru successfully steals back the shrink ray. The girls then suggest a day at a theme park. Gru agrees, believing he can abandon the girls there; but as he cannot, he begins to bond with them instead. Later, Gru contacts Perkins via video chat, stating that he finally has the shrink ray. Margo, Edith, and Agnes interrupt the meeting, and Perkins announces that he has lost confidence in Gru and will no longer fund his operations. As Gru tells the Minions he can no longer pay them for their services, the girls offer the contents of their piggy bank. With his Minions and himself inspired, Gru sells parts of his lair and the items he stole over the years to construct a spacecraft. Gru plans to steal the Moon when it is nearest to Earth, but this is the same day as the girls’ ballet recital. As Gru becomes conflicted, Dr. Nefario arranges for the girls to be returned to the orphanage, fearing the recital could ruin the plan. At the same time, Perkins informs his son Vector of Gru’s possession of the shrink ray and the adoption of the three girls, encouraging Vector to take action.
Gru successfully shrinks and steals the Moon. He rushes back to Earth to attend the recital, only to find a ransom note from Vector, who has infiltrated and abducted the girls. After arriving at Vector’s base, Gru surrenders the Moon. However, Vector procrastinates on the deal and after failing to stop Gru’s advance, flies off with the girls and the Moon. Meanwhile, Dr. Nefario has discovered that the effects of the shrink ray are temporary: the bigger the object is, the faster it reverts to its original size. Gru, Dr. Nefario, and the Minions execute a daring mid-air rescue of the girls, just before the Moon destroys Vector’s spaceship and launches itself back into orbit, along with Vector on it.
Sometime later, Gru re-adopts the girls, writing them a bedtime storybook based on his own experience. The girls perform their ballet recital for Gru, his mother Marlena, Dr. Nefario, and the Minions, only for one of the Minions to switch the music to something more modern, ending the film in a dance party.