The Sleepy Kittens scene
I was rewatching this movie on DVD the other day and was struck by the scene when Gru reads the Sleepy Kittens book to the girls, finishes, and looks up with the strangest look on his face. I forget what the last line of the book is exactly but it goes something like "for though while they sleep they are apart, their mother loves them with all their heart." Gru reads this line, his voice pausing ever so lightly, and then looks up in silence with an expression of sadness and confusion on his face, his lips parting slightly but soundlessly as if at a loss for words. A second later he jumps up, declining Agnes' request for good night kisses as he leaves the room.
This sweet little scene really adds a lot to Gru's character IMO. Kudos to the animators for portraying such a poignant moment. Given Gru's mommy issues, I can only imagine what could have been running through his head as he read that last line. Parenting is a huge theme in the movie and Gru is not only a parent but also a child, a "parentee" of his mom and the film tells the story not only of how he learns to be a dad but also how he overcomes his relationship with his mother.
Seeing this scene made me appreciate it even more when the director brought it full circle at the end where Gru's mom finally tells him that she's proud of him. Not for flying to the moon, not for saving the earth, but for becoming a dad. That was his greatest accomplishment of all. And of course the final shot of him and the three girls silhouetted against the moon was very sweet. :)