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- James Henry Trotter – The protagonist of the book, James is a seven-year-old orphaned boy who is forced into the care of his repulsive and abusive aunts, Spiker and Sponge, after his parents are killed by a rhinoceros. He wants nothing more than to have friends and be happy, which his aunts deny him. Also, James has been forced into slavery by Spiker and Sponge, who made James do all the work with no pay. James's wish is eventually granted, however, in the form of the companions he meets in the giant peach. By the end of his adventure, he gets more than he wished for in the form of millions of playmates in New York City. Something of a dreamer, James is, nonetheless, clever and ever-resourceful throughout his adventure in the giant peach, and his intuitive plans save his and his friends' lives on more than one occasion.
- The Old Man – A friendly yet mysterious wizard who is only seen once, yet is ultimately behind all of magical occurrences in the book, and also starts the adventure when he gives James a bag full of magical gems (crocodile tongue). It is these magical items which enchant the giant peach and its inhabitants, allowing James to begin his surreal journey and escape his evil aunts in the process. The wizard is not seen again after his encounter with James. However in the 1980 re-printing of the book, with illustrations by Nancy Ekholm Burkert, the mysterious old man can be seen in the final illustration hiding amongst the New York City crowd.
- Aunt Spiker – A dominating, cruel, malicious, and thoroughly repulsive lady, assumably the older of the aunts, who derives a sadistic pleasure in manipulating and enslaving young James, who she and her sister Sponge see as nothing more than a slave. Spiker is described as tall and thin – almost emaciated – with steel glasses. Both she and Sponge are vain, each singing praises of their imagined beauty while they are in fact repulsive, but each attacks the other's repulsiveness. James never hears either Aunt laugh out loud during his three years with them. Spiker meets her end when she is crushed to death as the giant peach rolls over her.
- Aunt Sponge – A greedy, selfish, and morbidly obese woman, and equally as cruel and repulsive as her older sister Spiker. Sponge is more gluttonous, thinking of eating the peach while Spiker seizes upon the money-making opportunities it will bring. Sponge is more or less dominated by Spiker, but attempts to save her own life instead of Spiker when she sees the giant peach rolling towards her. Nonetheless they trip up over each other and meet the same end.
- The Centipede – A male centipede, depicted as a boisterous rascal with a good heart, he is perhaps James' closest friend in the peach, taking an almost brotherly role to the boy. He is generally optimistic and even brave yet also loud-mouthed and rash, which gets himself and his companions into some bad situations, but his powerful jaws also save them on a few occasions. It was the Centipede who set the peach in motion by biting through the stem which connected it to the peach tree. The Centipede has an ego for many things including being the only actual pest of the group and his number of legs (he claims to have a hundred, he actually has only forty-two). He often asks for help with putting on his many boots, or taking them off, or shining them. In the last chapter of the book and after the destruction of the peach, it is revealed that he becomes Vice-President-in-Charge-of-Sales of a high-class firm of boot and shoe manufacturers.
- The Earthworm – An earthworm who hates the Centipede. Their arguments probably stem from the fact that they are the very opposite in temperament- the Centipede is very optimistic, whereas the Earthworm always expects the worst. The Earthworm is paranoid and has an extreme phobia of birds. He is also blind, and often imagines that things are worse than they really are. The Earthworm does however become an unwitting hero when he begrudgingly saves himself and the other inhabitants of the peach. They use him as bait to lure in over five hundred seagulls, which are then tied to the stem and used to hoist the peach out of the sea and away from sharks. The Earthworm is not without a warm, affectionate side; he is seen to get along well with James.The Earthworm becomes the mascot for a skin-cream firm when they reach New York.
- The Old Green Grasshopper – A male grasshopper, his personality has aspects of both the Centipede and the Earthworm, although he is generally more sophisticated (and certainly more optimistic than the Earthworm). The Old Green Grasshopper takes something of a fatherly role to James and is depicted as elderly, although he loves life more than the rest of the inhabitants of the peach and is a passionate musician, playing a violin from his own legs and providing music for his companions. In the last chapter of the book and after the destruction of the peach, it is revealed that he becomes a member of the New York Symphony Orchestra where his playing is greatly admired.
- The Ladybug – A kind, motherly female ladybug who takes care of James as if he were her son. She explains that the more black spots a ladybug has on the red shell, the more respectable and intelligent they are, and having nine spots, she is therefore very respectable and intelligent. In the last chapter of the book and after the destruction of the peach, it is revealed that the Ladybug, who had been haunted all her life by the fear that her house was on fire and her children all gone, married the head of the New York City Fire Department and lived happily ever after with him.
- Miss Spider – A good-natured female spider who takes care of James, taking an aunt-like role to the boy. Generally friendly and decent in manner, described by Dahl as having "a large, black and murderous-looking head, which to a stranger was probably the most terrifying of all". She has particular resentment towards Spiker and Sponge – especially Sponge, who is responsible for the cruel deaths of Miss Spider's father and grandmother. Miss Spider makes hammocks using her webs for the others to sleep in. Her webs are very strong and it is her webs, along with silk from the Silkworm, which tie the flock of seagulls to the stem of the giant peach and enable it to be lifted out of the sea and into the air, escaping the sharks.
- The Glowworm – A female glowworm, she quietly hangs from the ceiling in the hollowed-out stone at the center of the giant peach and provides lighting for the interior of the fruit in the form of a bright green bioluminescence. An incessantly sleepy character, she doesn't speak often and is slow to move. After the adventure, she saves New York from an enormous electric bill by illuminating the Statue of Liberty's torch.
- The Silkworm – A female Silkworm. Often asleep, a possible reference to hibernation, she helps Miss Spider to make ropes for the seagulls. However, she has no dialogue.
- Cloud Men – Are minor antagonists, who threw rocks and supplies at the peach after Centipede taunts them.
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