The attraction opened in 2012 but will stop service next year due to the expansion of the Avengers Campus.
Construction is set to begin in early 2025, leading to the removal of the Red Car Trolley's barn.
The iconic vehicles are modeled after the Pacific Electric Railway trolleys that characterized the streets of Los Angeles for nearly 75 years.
The removal comes after Disney announced its plans to double its current Avengers themed-land size with the addition of two new rides.
Stark Flight Lab will immerse guests inside Iron Man's workshop, while Avengers Infinity Defense will invite riders to take on Thanos, as announced at D23, Disney's fan event.
The confirmed date for the trolley's closure will be announced later this winter.
Other newly announced theme lands and rides are coming to Disneyland, including Tiana's Bayou Adventure this Fall, a "Coco" inspired ride and a new "Avatar" experience.