On the bank of a tranquil river, a girl called Alice grows bored of listening to her older sister read aloud from a history book of William I of England. When her sister chastises Alice's daydreaming, Alice tells her kitten Dinah that she would prefer to live in a nonsensical dreamland called Wonderland. Alice and Dinah spot a waistcoat-wearing White Rabbit passing by, and Alice gives chase as he rushes off crying that he is "late for an important date". Alice follows him into a rabbit hole and falls into a labyrinth. Her dress catches her fall like a parachute and she begins to float, then she floats upside down and her feet catch on a bar to break her fall. She sees the White Rabbit disappear into a tiny door and tries to follow, but the door's talking knob advises her to alter her size using a mysterious drink and food. She drinks from a bottle marked "Drink Me", and shrinks down to the proper size to open the door. But when Alice learns the door's locked and she's too small to reach the key atop the table, a box of "Eat Me", "Try Me", and "Take One" cookies (cakes) appear. But once Alice takes a bite, she grows into a giant, filling up the entire room. She then begins to weep large tears that splash like big puddles, causing the room to flood. The doorknob then tells Alice there's a little liquid left in the bottle, so she stops crying, drinks the last drop and eventually manages to shrink into the bottle and passes through the door's keyhole and into Wonderland. She meets several strange characters including the Dodo and Tweedledee and Tweedledum who recount the tale of "The Walrus and the Carpenter".
Alice eventually finds the White Rabbit in his house, but before she can ask what he is late for, she is sent to fetch some gloves after being mistaken for his housemaid. She eats a cookie and grows into a giant again, getting stuck in the rabbit's house. She tries to pull herself out, but is too big. The White Rabbit, the Dodo, and chimney sweep Bill the Lizard believe Alice to be a monster and plot to burn the house down. Alice escapes by eating a carrot and shrinking down to the size of an insect. She meets and sings with some talking flowers, but they chase her away upon accusing her of being a weed. Alice is then instructed by the hookah-smoking Caterpillar to eat a part of his mushroom grow back to her original size. Alice decides to keep the remaining pieces of the mushroom on hand.
Alice meets the Cheshire Cat who advises her to visit the Mad Hatter, March Hare and the Dormouse. The three are hosting a mad tea party and celebrate Alice's "unbirthday", a day where it is not her birthday. The White Rabbit appears, but the Mad Hatter and the March Hare destroy his pocketwatch and throw him out of the party. Fed up with all the wonderlandians' rudeness and wackiness, Alice abandons her pursuit of the White Rabbit and decides to go home, but gets lost in the Tulgey Wood. The Cheshire Cat appears and leads Alice into a giant hedge maze ruled by the tyrannical Queen of Hearts and her smaller husband, the King of Hearts. The Queen orders the beheading of anyone who enrages her, and invites Alice in a bizarre croquet match using flamingoes and hedgehogs as the equipment.
The Cheshire Cat appears again and pulls a trick on the Queen which she accuses Alice of doing, and Alice is put on trial. Just then, she remembers that she still has the remains of the Caterpillar's mushroom. She eats it and grows to an enormous height which the King claims is forbidden in court. Now a gigantic size, Alice feels free to speak her mind and in doing so she openly insults the Queen. However, she had hastily eaten both sides of the mushroom and shrinks to her normal size. She is forced to flee after the Queen orders her execution. Alice becomes pursued by most of Wonderland's characters until she finally reunites with the Doorknob, who then tells her she is having a dream, forcing Alice to wake herself up. The film ends as Alice, her sister and Dinah head home for tea.
- In the Walrus and the Carpenter sequence, the R in the word "March" on the mother oyster's calendar flashes. This alludes to the old adage about only eating oysters in a month with an R in its name. That is because those months without an R (May, June, July, August) are the summer months in England, when oysters would not keep due to the heat, in the days before refrigeration.
- The Doorknob was the only character in the film that did not appear in Lewis Carroll's books.
- HIDDEN MICKEY: In the scene where Alice grows and gets stuck in the White Rabbit's house, if you look closely at the DoDo bird's flame as he lights his pipe, there is a hidden Mickey flickering in the flame.
- In the Walrus and the Carpenter sequence, the dates on the calendar are the same as they would be in March 2010, when Disney would release Alice in Wonderland (2010), directed by Tim Burton.
- An earlier adaptation was planned for the thirties. The storyboards were done by the talented British artist David Hall. It was a bit closer to the book, but it was rejected for being too scary. Amongst the concepts from this version was the Mad Hatter and March hare chasing Alice with a knife and scissors, the Cheshire Cat with hundreds of sharp teeth, and Alice nearly beheaded by a grinding gear.
- In the song "Painting the Roses Red", after Alice sings "Oh, pardon me, but Mister Three, why must you paint them red?" the three cards say "Huh? Ohhhhhhh!". When they say that, The three of clubs card has a three in the bottom right corner with no clubs symbol on top of the three. the three of clubs also has nothing in the top left corner. The ace and two of clubs have nothing in both of their corners. A few slides later, the symbols in the corners come back. (Possibly a chameleon-ability similar to the Caterpillar.)
- When Alice is crawling out of the White Rabbit's house after being shrunk, the door is shut. When she runs to the step, the door is open.