When Lucy is tazing Gru he drops his freeze ray but when it shows her trying to pick him up it is gone.
When we are first introduced to Eduardo/El Macho, he orders cupcakes with Mexican flags on them. He then opens his shirt to display a tattoo of the Mexican flag. Towards the end of the movie when he his wearing the El Macho villain suit, most of his chest is visible and the tattoo is no longer there.
At the start of the film, when the secret lab is stolen from the Arctic, the footage is being viewed in AVL headquarters. The information on their screen includes latitude and longitude co-ordinates.
These co-ordinates are actually in southern Sudan, rather than the Arctic circle.
When Lucy is in the cake shop and smashes up the cup cakes she never gets any of the frosting on her hands. This occurs even when she smears it over Gru's face.
When Gru and Lucy break into Salsa and Salsa, the booby trap line is not visible before nor after Gru trips it.
Early in the movie one of the minions is vacuuming and answers the front door. The door opens in. Later when Gru is sitting on the front porch and Agnes comes out with an umbrella, the door opens out.
When Dave comes out of the kitchen in the cupcake shop with the minion cupcake, Lucy splits it apart, then pins Dave down and asks Gru if he knows him. But when she captured Gru and took him to the submarine, there were two minions with him and Lucy never asked about them then.
At the beginning of the film, El Macho's ship first attracts the coins of the two card players. This is impossible as globally, all coins are minted using non-ferrous metals or alloys so as not to be magnetizable.
When Gru pops out of Lucy's trunk at the AVL he has a starfish on his head, this would imply 1) that there was a large enough gap in the trunk for the starfish to get in and therefore Gru to escape and 2) that water was getting into the trunk, and Gru would have drowned in the time it took to get to the AVL.
During the fight scene after they exit Eduardo's place, Eduardo's eyes are clear of that gunk that was sprayed on him to blind him.
When Gru is trying to enter El Macho's secret lair, a blob of guacamole falls on one of the tiles, causing an ax to fall. The guacamole disappears for the remainder of the scene.
After El Macho finishes playing la cucaracha on the floor tiles so he can enter his secret lair, he ends 2-3 tiles from the entrance. There's a cut to a close-up of El Macho and he is only 1 floor tile away from the entrance.
Eduardo's pet chicken, Pollito, is being referred to by male pronouns, even though "he" is clearly a "she", as he lacks the big tail of roosters.