The cartoon follows Mickey Mouse and his dog Pluto traveling by train, avoiding a rule forbidding dogs; Pete plays a menacing conductor intent on enforcing the rule.
Leaving for vacation, Mickey Mouse and Pluto arrive at a train station in Burbank, California (home of Walt Disney Productions headquarters). They board a west-bound train, but are both immediately kicked off by the conductor, played by Pete, that dogs aren't allowed (side gag reveals Pluto's luggage to contain bones). Pete then rambles off the train's destinations and forces his watch to tell him when the train is ready to leave. When the watch does show its time for the train to go, Pete calls "All aboard!"
Mickey, at this point, decides to smuggle Pluto onboard by squeezing the dog inside his suitcase. At first, the handle breaks, making Mickey almost leave Pluto behind, but he recovers the suitcase and manages to make it aboard the caboose just as the train is leaving the platform. Later, Pluto barks, wanting to be let out. Mickey reminds him that he will be thrown off if Pete finds out that Pluto's on the train, so he takes Pluto out and unsquashes him. The freedom is only short lived, however, as Pete is coming through the train to collect tickets, forcing Mickey to squash Pluto back into position in the suitcase. After biting "OK" in Mickey's tickets, Pete sees Mickey's suitcase containing Pluto in the seat and forcefully throws it into an overhead baggage net. This causes Pluto to bark, making Pete suspicious.
He then recognizes Mickey, who tries to hide behind a large newspaper and make it look like the barking was coming from him. Realizing that Pluto has been stowed in the suitcase, Pete menacingly asks Mickey if he is alone, then makes up a story about owning a little cat who'd cry when he was all alone and screams a loud "MEOW!" (With his face closely resemble a real cat) at the suitcase causing Pluto to leap out. Realizing his cover's been blown, Pluto ducks back into the suitcase, but Pete has already figured it out. Before he can catch the pair, Mickey and Pluto run away and a chase ensues on board the train.
Mickey and Pluto first hide in a sleeping car where Pete mistakenly intrudes on a female passenger and gets assaulted. Pete then stumbles into another bed where Mickey and Pluto (disguised as babies) are hiding and when he grabs them, they beat him. Pete apologizes for the intrusion, but quickly catches on after realizing he was covering up Pluto's tail. Just as Mickey and Pluto are gloating that they'd fooled Pete, Pete bursts in and threatens to beat them to a pulp, but sudden darkness from the train running through a tunnel allows the pair to escape, leaving Pete to beat the mattress to a pulp and experience a brief entanglement with the springs.
Mickey and Pluto masquerade as a conductor by hiding in Pete's own coat and hat which they'd had taken off in the tunnel. After getting false directions from Mickey, Pete catches on and threatens to catch them, but ends up disturbing the female passenger again. Pete receives another beating and, unintentionally taking the passenger's hat, gets pricked by one of her needles. Mickey disguises himself as an Indian chief with Pluto in his papoose, but Pete sees through their disguise after Pluto bites his hand.
While Mickey and Pluto are next to an open window, Pluto is caught on a passing mail hook which whisks him outside the train. Mickey runs after him through the train, and is just able to grab Pluto as he exits the caboose. Pete throws their luggage out after them and they fall to the ground from the mail hook. Mickey then looks up at the station sign and is pleasantly surprised that they have arrived at their destination, Pomona.
Mr. Mouse Takes a Trip is unique among the classic Disney shorts in that film footage exists of the voice-over session, which included Walt Disney and Billy Bletcher. According to film historian Leonard Maltin, the footage was not known to exist and only discovered (as of 2004) "not too many years ago."
The black-and-white film, which is about ten minutes in length, is the only known footage of Disney performing as Mickey Mouse and was included on the 2004 DVD release "Walt Disney Treasures: Mickey Mouse in Living Color, Volume Two" as an easter egg. Edited portions of the footage were also seen in earlier releases.
In September 1940, a one-page adaptation of Mr. Mouse Takes a Trip appeared in Good Housekeeping magazine. In this version Pete discovers Pluto by seeing his tail sticking out of the suitcase. Instead of being kicked off the train, Mickey and Pluto are confined to the baggage car. The story was told in verse and was illustrated by Tom Wood.
In October 1940, a prose version of Mr. Mouse Takes a Trip was printed in the first edition of Walt Disney Comics and Stories. This five-page version is a closer retelling of the film, with the added detail that Mickey is heading to an "important meeting" in Pomona which he can't be late for. Pluto comes along only because he would get lonely if he stayed home alone.
In 2010, the film inspired the Italian comic story "Topolino, Pluto e la gita in montagna," or "Mickey, Pluto, and the Trip to the Mountain." The story, published in the May edition of Extralarge XL Disney, is 25 pages and written and illustrated by Enrico Faccini1940 – Original theatrical release
c. 1965 – Highlights included in "Mickey's Memorable Moments, Volume 2" (Super 8)
c. 1980s – "A Disney Vacation" (TV)
1984 – "Cartoon Classics - Limited Gold Edition: Mickey" (VHS)
1986 – "Adventures with Mickey" (TV)
1998 – The Ink and Paint Club, episode # 1.43: "On Vacation" (TV)
1998 – "The Spirit of Mickey" (VHS)
2004 – "Walt Disney Treasures: Mickey Mouse in Living Color, Volume Two" (DVD)
2006 – "Walt Disney's Funny Factory: With Mickey" (DVD)
2009 – "Walt Disney Animation Collection: Classic Short Films Volume 1: Mickey and the Beanstalk" (DVD)
2009 – Have a Laugh! (TV)
2010 – "Have a Laugh!: Volume 2" (DVD)