The San Diego Trolley Green Line is the newest trolley route. First entering service on July 10, 2005 upon the opening of the 5.9 mile Mission Valley East Extension. Service originally ran between Old Town and Santee, with trains servicing 4 new stations as part of this extension. The opening of the Green Line saw changes to other trolley routes as well, with the Green Line replacing the Blue Line at 8 stations previously served by the Blue Line (from Old Town to Mission San Diego), and 1 station previously served by the Orange Line (Santee), with Green and Orange Line trains sharing 5 stations.

The service change (and not just extending the Blue Line) and the lack of Green Line service to downtown was done because certain stations on the new extension (I believe the new underground SDSU station in particular) required trolley service with new low-floor S70 trolleys for accessibility, which required 6 inch height platforms, which the original stations opened with the Green Line were retrofitted for, but not the rest of the trolley that only had 4 inch or ground-level platforms. This meant all Green Line trains were generally two trolleys long, one low-floor S70 (these original low-floor trolleys are longer at 90 feet instead of 80 feet long) and one high-floor SD-100.

In 2012 the first phase of the Trolley Renewal project was completed, with all stations between Old Town and 12th & Imperial rebuilt given an extra two inches of platform height and allowing the Green Line to now run through to Downtown, with trains extended south via the Old Town and through downtown via the Bayshore Line. With this service change the Green Line was extended to an additional 10 stations, taking over service from stations originally served by the Blue Line (which was re-extended to Old Town and north to UTC, with the opening of the Mid-Coast trolley extension in 2021) and the Orange Line. This service change was also made possible by the delivery of new S70 US streetcars (these are 10 feet shorter than the original S70 cars, so a three-car train can fit within downtown San Diego City blocks).

From 2017 the Sycuan Casino sponsered the Green Line in a deal that was originally supposed to last for up to 30 years and generate up to $25.5 million in revenue for MTS operations. This naming rights deal ended at some point during the 2020 COVID-19 Pandemic with all references to the Sycuan removed from MTS literature and Green Line signs during a 2022 San Diego Visit.