Miss Spider, as a giant, speaks with a French accent and wears a beret and a scarf. She is soft-spoken, brave, tough, feisty, kind, strong, and confident. She is like a mother figure to James because he saved her from being killed by his cruel aunts. She initially preferred to be alone, but later warms up. Her love interest is Mr. Centipede, who often tries her patience, but she soon falls in love with him.
She is first seen as an ordinary spider in James Henry Trotter's window. He tells her it is a bad idea to build her web there because his aunts might see her and try to kill her. He introduces himself to her and expresses his desire to go to New York and shows her a special candle his parents gave him.
The next morning, Aunt Spiker and Aunt Sponge spot her, but James saves her, carries her outside, and tells her to leave as fast as she can. That night, James climbs inside the peach, having been transformed, and Miss Spider, who has been magically transformed into a giant along with the other bugs, assures him that no one will hurt him, although he does not recognize her.
During their journey to New York City, she aids in a dangerous and desperate situation in which the crew on the peach have to fend off a robot shark. Later on, the bugs and James decide to eat some of the peach so they won't be so hungry on their journey to New York, an event that Miss Spider chimes in on. That same night, when she is tucking the boy into bed using her web and his coat, Miss Spider gets James to realize that she is the same spider whose life he had saved at his aunts' house. It is at this moment that James understands why she has cared for him so much during the journey. After she's done wrapping the boy up with her webbing, Miss Spider comforts James when he is confronted by the thoughts of his tortured past and kisses him goodnight, which is a further confirmation that she is like a mother to the boy now.
She later starts a romantic relationship with Centipede after he saves her and James from some Skeleton pirates. Although she and the others are separated from James following an attack from the Rhino, they reunite with him and aid him in the capture of Spiker and Sponge, who had journeyed to New York to get James and the peach back. Miss Spider binds James' aunts up in her webbing, and they are taken away. She then lives in Central Park with James and the others, and becomes the owner of "Spider Club."
- During the set of when Miss Spider is normal-sized, Paul Terry (who played James) got bitten by the spider. This is why he never acted again.
- She is one of the few female Disney characters to be seen smoking a cigar (though she actually borrowed it from Mr. Centipede).
- In the book, she, along with the Silkworm (character in book), set up a factory to make ropes for tightrope walkers in New York City.