Two hunters (Jack Lester
, Kenneth Jennings
) ride through the forest searching for game when they come to a quiet part of the forest. One hunter announces that the forest is magical; hence why there is no snow that falls and it always seems like spring, and that there must be a unicorn living there. The other hunter is skeptical and claims that there are no more unicorns left in the world. The other argues that there must be just one left and, while she exists, there will be no game to hunt in this forest. They turn around, watched by a unicorn (Mia Farrow
) who wonders if she is indeed the last. She is approached by a butterfly (Robert Klein
) who sings to her and quotes poetry; a typical trait of most butterflies. She asks him if, in all his travels, he's ever seen another unicorn. He taunts her, at first, with riddles and nonsensical lyrics before announcing that all the other unicorns were taken away by a Red Bull that covered their tracks long ago. He then flies away, singing. The unicorn, though hesitant, resolves to leave the safety of her forest and search for the others.
She travels over hundreds of miles before coming to a series of farmlands. One elderly farmer (Jack Lester
) tries to tame her, mistaking her for a white mare; a mistake which infuriates the unicorn. She cannot believe that all the man sees her as is but a horse and she runs off. However, she comes to realize that if man has forgotten what a unicorn looks like, there may very well be unicorns left in the world hiding in plain sight. One night, while she's sleeping, a side-show caravan led by the witch Mommy Fortuna (Angela Lansbury
) comes along. Fortuna places a spell on the unicorn to keep her sleeping before putting her in a cage. The next evening, Fortuna's lackey, Ruhk (Brother Theodore
), entertains a group of spectators, introducing them one by one to Fortuna's freaks of nature. He finally shows them the unicorn who has a fake horn placed on her head so that they may actually see it. She is approached by Fortuna's magician, Schmendrick (Alan Arkin
), who can see her for what she is and asks her to point out the other captives of the side-show. The unicorn reveals most of them to be normal animals with illusions placed on them, all but the harpy, Celaeno (Keenan Wynn
), who is real and as immortal as the unicorn.
That night, Schmendrick uses his somewhat faulty magic to free the unicorn who then, in turn, frees all the other captives. However, when she frees the harpy, it turns and attacks her. Mommy Fortuna comes outside with Ruhk to investigate the ruckus and they are both killed by the vengeful harpy while the unicorn and Schmendrick escape. Though Fortuna predicted her death by the claws of the harpy, she dies deliriously happy that the harpy will always know that Fortuna was the one to cage her. Schmendrick accompanies the unicorn on her quest to find the other unicorns and tells her that he knows of a Red Bull that is owned by a king named Haggard (Christopher Lee
), though his very existence is shrouded in mystery. Before very long, a group of bandits ride down the road and, while the unicorn hides, Schmendrick is taken captive. He's brought back to their camp where he's welcomed by their leader, Captain Cully (Keenan Wynn
) and the scullery maid, Molly Grue (Tammy Grimes
). Schmendrick surprises them all, and himself, with a bout of successful magic in which he procures images of the legendary Robin Hood and his merry men. Irritated and frightened by Schmendrick's abilities, Captain Cully ties him to a nearby tree. Schmendrick tries to free himself with magic but accidentally brings the tree to life. The tree (Nellie Bellflower
) falls in love with him and nearly smothers him until the unicorn arrives and uses her magic to free Schmendrick. They are then joined by Molly Grue who can also see the unicorn for what she is and weeps at the sight of her, cursing her for coming to her as an older woman rather than a young maid. Molly joins the group as they continue on their journey, much to Schmendrick's disapproval.
When they finally have Haggard's castle in sight, the group is suddenly attacked by the Red Bull which ignores Schmendrick and Molly and attempts to herd the unicorn towards the castle. Urged by Molly to do something, Schmendrick uses a great deal of magic to transform the unicorn into a human girl. Confused by the unicorn's different form, the Red Bull leaves. Molly criticizes Schmendrick for turning the unicorn into a mortal and the unicorn is equally upset, crying out that she can 'feel this body dying all around me.' Nevertheless, they continue to the dreary castle and are welcomed into Haggard's decrepit hall, introducing themselves as Haggard's new magician and cook accompanied by the Lady Amalthea. Intrigued by Schmendrick's potential to please him, Haggard dismisses his current magician, Mabruk (Paul Frees
), who laughs at Haggard's ill judgement, claiming that he's let his doom walk right through the front door. Lady Amalthea catches the eye of both Haggard and his adopted son, Lir, (Jeff Bridges
), though she ignores the advances of both and spends her days wandering the castle, slowly losing memory of who she was or what she was looking for.
Realizing that they are losing time and Amalthea is becoming more and more human, Schmendrick and Molly do their best to discover where the lair of the Red Bull is while performing daily tasks to keep Haggard entertained. Finally, Molly receives word from the resident cat (Don Messick
) that the lair is located at the base of an old clock, but will only grant entry at the right 'time'. Molly scorns the cat for speaking only in riddles, but the cat merely admits that no cat ever gave a straight answer. Meanwhile, Amalthea and Lir fall in love while Haggard suspects her of her true nature. He tells her that he once saw a unicorn and that it gave him such joy that he sought to keep it for himself. So he commissioned the Red Bull to gather up all the unicorns of the world and drive them into the sea below his castle so that he may look upon them every day.
Molly tells Schmendrick that she knows where the entrance to the Bull's lair is and they gather Amalthea and head to the old clock in the main hall. Across from the clock, upon a bureau, lies a crowned skeleton who speaks to them but can only giggle and laugh at their frustration. Per instructions from the cat, Molly produces a bottle of water; the best she can do in lieu of the wine she was supposed to get. When Schmendrick feigns turning the water into wine, the skeleton's interest peaks and he agrees to tell them the secret of entering the lair if they give him the 'wine', now an empty bottle. The skeleton tells them that all they have to do is walk through the clock before gulping down the invisible liquid. However, when he catches sight of Amalthea, he recognizes her as a unicorn and cries out to Haggar. Haggar runs down to the hall and swings his sword at Schmendrick just as Molly and Amalthea enter the realm between the clock and the lair. Schmendrick soon follows just as Haggar destroys the clock portal, trapping them inside. Lir appears, stating that he saw where Amalthea went and he followed.
All together, they walk down the lair in search of the exit. Schmendrick explains their plight to Lir who reveals that he doesn't care who or what Amalthea is; he loves whom he loves. Amalthea is inspired by his words and claims that she wants to remain human and die with him, but Lir objects. He tells her that, as a hero, he knows when things should happen, and what should happen is that Amalthea continue her quest and save the unicorns, even if it means that she must become one again and therefore be unable to return Lir's love. The Red Bull suddenly appears and recognizes Amalthea for what she really is. Schmendrick manages to transform her back into a unicorn and she leads the Bull out of its lair onto the beach. With Schmendrick unable to do anything, Lir steps forward but is trampled by the Bull. Enraged, the unicorn turns to face the Bull and intimidates it into the sea where a large wave appears, unicorns emerging from the foam. They envelope the Bull in sea water before storming the beach. They all run upwards towards the castle, releasing it from its rocky base. Before he tumbles to his death in the sea, Haggar shouts out to the unicorn, "I knew you were the last!"
The unicorn returns to Molly and Schmendrick and revives Lir before saying her farewells and departing. The remaining trio returns to the countryside where Lir says his goodbyes before leaving to find his own way. Schmendrick asks Molly to go with him and she agrees. Later the next evening, as they sleep at their camp, the unicorn returns to Schmendrick and expresses her thanks, even if she is the first unicorn ever to feel the sting of mortality and the pain of regret. Schmendrick apologizes but the unicorn tells him that she also learned love, and there are unicorns in the world again thanks to him. For that, she is grateful even if she must feel regret for leaving. She returns home to her forest as Molly and Schmendrick look on after her; at the last unicorn.