Mrs. Ladybug is a kindhearted, middle-aged ladybug who becomes an aunt figure to James, and hates rudeness. She was once an ordinary ladybug, but became life-size when James' peach became giant. While she is the good bug she is, she can also be firm, tough, feisty, and yet brave. This is proven when she fends off the robot shark's torpedoes with her purse when they attack James and Mr. Grasshopper.
She first appears along with the other bugs when James climbs inside the peach and assures him that he's safe and shows him that he has changed as well as all of them. She accompanies the others on their journey to New York City and even helps them defeat a robot shark that attacks the group. After they reach New York, she and the other bugs are separated from James during an attack from the Rhino, but they later reunite with James, who has by then, resumed his normal form. She helps in the capture of Spiker and Sponge and, along with James and the other bugs, is joyfully welcomed by the people of New York. She, James, and the bugs get to live inside the pit of the peach in Central Park after they let the people eat the peach. They all live happily ever after, and Mrs. Ladybug becomes a nurse.