In Shostka, Russia, in 1885, the Mousekewitzes, a Russian-Jewish family of mice who live with a human family named Moskowitz, are having a celebration of Hanukkah where Papa gives his hat to his 7-year-old son, Fievel, and tells him about the United States, a country where he believes there are no cats. The celebration is interrupted when a battery of Cossacks ride through the village square in an anti-Jewish arson attack and their cats attack the village mice. Because of this, the Moskowitz home, along with that of the Mousekewitzes, is destroyed, while Fievel has a narrow escape from the cats. They flee the village in search of a better life.
In Hamburg, Germany, the Mousekewitzes board a tramp steamer setting sail for New York City. All the mice aboard are ecstatic at the process of going to America as there are "no cats" there. During a thunderstorm on their journey, Fievel suddenly finds himself separated from his family and washed overboard. Thinking that he has died, they proceed to the city as planned, though they become depressed at his loss.
However, Fievel floats to New York City in a bottle and, after a pep talk from a French pigeon named Henri, embarks on a quest to find his family. He is waylaid by conman Warren T. Rat, who gains his trust and then sells him to a sweatshop. He escapes with Tony Toponi, a street-smart Italian mouse, and they join up with Bridget, an Irish mouse trying to rouse her fellow mice to fight the cats. When a gang of them called the Mott Street Maulers attacks a mouse marketplace, the immigrant mice learn that the tales of a cat-free country are not true.
Bridget takes Fievel and Tony to see Honest John, an alcoholic politician who knows the city's voting mice. However, he can't help Fievel search for his family, as they have not yet registered to vote. Meanwhile, his older sister, Tanya, tells her gloomy parents she has a feeling that he is still alive, but they tell her to let go of these feelings as it doesn’t seem possible that Fievel could still be alive.
Led by the rich and powerful Gussie Mausheimer, the mice hold a rally to decide what to do about the cats. Warren is extorting them all for protection that he never provides. No one knows what to do about it, until Fievel whispers a plan to Gussie. Although his family also attends, they stand well in the back of the audience and they are unable to recognize Fievel onstage with her.
The mice take over an abandoned museum on the Chelsea Piers and begin constructing their plan. On the day of launch, Fievel gets lost and stumbles upon Warren's lair. He discovers that he is actually a cat in disguise, and the leader of the Maulers. They capture and imprison Fievel, but his guard is a reluctant member of the gang, a goofy, soft-hearted long-haired orange vegetarian tabby cat called Tiger, who befriends and frees him.
Fievel races back to the pier with the cats chasing after him when Gussie orders the mice to release the secret weapon. A huge mechanical mouse, inspired by the bedtime tales Papa told Fievel of the "Giant Mouse of Minsk", chases the cats down the pier and into the water. A tramp steamer bound for Hong Kong picks them up on its anchor and carries them away. However, a pile of leaking kerosene cans has caused a torch lying on the ground to ignite the pier, and the mice are forced to flee when the human FDNY arrives to extinguish it.
During the fire, Fievel is once again separated from his family and ends up at an orphanage. Papa and Tanya overhear Bridget and Tony calling out to Fievel, but Papa is sure that there is another "Fievel" somewhere, until Mama finds his hat.
Joined by Gussie, Tiger allows them to ride him in a final effort to find Fievel and they are successful. Papa returns Fievel’s hat, commenting that it now fits him and he has grown up into a mouse. The journey ends with Henri taking everyone to see his newly completed project—the Statue of Liberty, which appears to smile and wink at Fievel and Tanya, and the Mouskewitzes' new life in the United States begins.
Phillip Glasser as Fievel Mousekewitz. While "Fievel" is a generally accepted spelling of his name, the opening credits spell it as "Feivel", the more common transliteration[4][5] of the Yiddish name (פֿײַװל Fayvl) (Cf. Shraga Feivel Mendlowitz and Feivel Gruberger). The main protagonist, he is the seven-year old only son of Papa and Mama Mousekewitz. A high-spirited, daring yet naïve Russian-Jewish mouse, Fievel becomes a frightened child when he is separated from his family. The strength and the encouragement from his new American friends from Henri to Tony and Bridget give him the fortitude to reach his goal in reuniting with his family while starting a new life in America. Fievel was the same name of Steven Spielberg's grandfather, whose stories as an immigrant influenced the movie (the ending credits spell his name as "Fievel"). However, many English-speaking writers have come to adopt the spelling Fievel, especially for this character; it was this spelling that was used on the film's poster, in promotional materials and tie-in merchandise, and in the title of the sequel An American Tail: Fievel Goes West. His last name is a play on the Jewish-Russian last name "Moskowitz", the name of the human occupants of the house his family is living under in the beginning of the film.
John Finnegan as Warren T. Rat, a small cat who disguises himself as a rat. Leader of the Mott Street Maulers, an all-cat gang which terrorizes the mice of New York City. Conniving and a schemer, he misleads the gullible Fievel at one point. Later on, he gets his comeuppance when Fievel discovers and later exposes him as a cat to the Mouse community. He is accompanied everywhere by his accountant Digit, a small British-accented cockroach.
Amy Green as Tanya Mousekewitz (singing voice provided by Betsy Cathcart), Fievel's 8-year-old older sister, whom he mutually adores. Optimistic and cheerful, less daring but more obedient than her brother, she alone believes (correctly) that he survived being washed overboard en route to the United States. She is given the American name "Tillie" at the immigration point at Castle Garden.
Nehemiah Persoff as Papa Mousekewitz, the head of the Mousekewitz family who plays the violin and tells stories to his children.
Erica Yohn as Mama Mousekewitz, Fievel's mother. Countering Papa's dreamy idealism, she is a level-headed pragmatist, besides being stricter with their offspring than he is. She also has a fear of flying.
Pat Musick as Tony Toponi, a streetwise teenage mouse of Italian descent. His "tough guy" attitude suits his New York surroundings. The name "Toponi" is a play on "topo", the Italian word for "mouse". He hits it off with Fievel, acting as a surrogate big brother to the younger mouse, whom he calls “Philly”. In a subplot, He falls in love with Bridget.
Dom DeLuise as Tiger, the most physically-imposing member of the Mott Street Maulers, whom he serves as an enforcer...and often as the brunt of their cruel jokes. This bushy-tailed, orange long-haired tabby stands 3 feet tall on his rear legs. Although not especially intelligent Tiger is very friendly, and his warm nature endears him to mice and birds. He is mostly vegetarian, aside from the occasional bit of fish. He enjoys card games like poker and gin rummy, despite being terrible at them. Tiger's voice also helps him stand out; he sings bass and baritone, from D2 or E2 to F4 or G4.
Christopher Plummer as Henri le Pigeon, a pigeon of French descent, who oversees construction of the Statue of Liberty.
Cathianne Blore as Bridget, an attractive, elegant Irish-born mouse and Tony's significant other. Her parents were slain and devoured by the Mott Street Maulers, making her an advocate in speaking out against the cats. Kind, passionate yet soft-spoken, she acts as a surrogate big sister to Fievel.
Neil Ross as Honest John, a local Irish-born mouse politician who knows every voting mouse in New York City. An ambulance-chasing drunkard, he takes advantage of voters' concerns to increase his political prestige. John is a caricature of the 19th-century Tammany Hall politicians.
Madeline Kahn as Gussie Mausheimer, a German-born mouse considered to be the richest in New York City, who rallies the mice into fighting back against the cats.
Will Ryan as Digit, Warren's British cockroach accountant who has a fondness for counting money, but is plagued by frequent electrical charges in his antennae whenever he gets nervous or excited.
Hal Smith as Moe, a fat rat who runs the local sweatshop. Fievel is sold to him by Warren.
Dan Kuenster as Jake, Warren's burly aide-de-camp. Among the Mott Street Maulers, he alone enjoys listening to his leader's violin music. Jake catches Fievel after a chase through the sewers. After Tiger takes pity on Fievel and sets him free, Jake and his fellow Maulers pursue the young mouse to the Chelsea Pier...only to face the "Giant Mouse of Minsk".