- Mole: A mild-mannered, home-loving animal, and the first character introduced. Fed up with spring cleaning in his secluded home, he ventures into the outside world. Initially overawed by the hustle and bustle of the riverbank, he eventually adapts.
- Rat: Known as "Ratty" to his friends (though actually a water vole), he is cultured, relaxed and friendly, with literary pretensions and a life of leisure. Ratty loves the river and takes Mole under his wing. He is implied to be occasionally mischievous and can be stubborn when it comes to doing things outside his riverside lifestyle.
- Mr. Toad: The wealthy scion of Toad Hall who inherited his wealth from his late father. Although good-natured, kind-hearted and not without intelligence, he is also spoiled, conceited, and impulsive. He is prone to obsessions and crazes (such as punting, houseboats, and horse-drawn caravans), each of which in turn he becomes bored with and drops. His motoring craze eventually sees him imprisoned for theft, dangerous driving and gross impertinence to the rural police. Several chapters of the book chronicle his daring escape from prison.
- Mr. Badger: Gruff and solitary, who "simply hates society", Badger embodies the "wise hermit" figure. A friend of Toad's late father, he is uncompromising with the disappointing Toad yet remains optimistic his good qualities will prevail. He lives in a vast underground sett, part of which incorporates the remains of a buried Roman settlement. A brave and a skilled fighter, Badger helped clear the Wild Wooders from Toad Hall with his large cudgel.
- Otter and Portly: A friend of Ratty with a stereotypical "Cockney costermonger" character, the extrovert Otter is tough and self-sufficient. Portly is his young son.
- The Weasels: The story's main antagonists. They plot to take over Toad Hall.
- Pan: A god who makes a single, anomalous appearance in Chapter 7, "The Piper at the Gates of Dawn".
- The Wayfarer: A vagabond seafaring rat, who also makes a single appearance. Ratty briefly considers following his example, before Mole manages to persuade him otherwise.
- Squirrels and rabbits, who are generally good (although rabbits are described as "a mixed lot").
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