The 1965 Giro d'Italia was the 48th running of the Giro d'Italia, one of cycling's Grand Tour races. The Giro started in San Marino, on 15 May, with a 295 km (183.3 mi) stage and concluded in Florence, on 6 June, with a 136 km (84.5 mi) leg. A total of 100 riders from 10 teams entered the 22-stage race, which was won by Italian Vittorio Adorni of the Salvarani team. The second and third places were taken by Italian riders Italo Zilioli and Felice Gimondi, respectively.

Ten teams were invited by the race organizers to participate in the 1965 edition of the Giro d'Italia. Each team sent a squad of ten riders, which meant that the race started with a peloton of 100 cyclists. From the riders that began the race, 80 made it to the finish in Florence.

The teams entering the race were:

From the 100 riders that started the Giro, only 10 were not Italian.

The race route was revealed to the public on 25 March 1965 by race director Vincenzo Torriani. San Marino hosted the start of the race, which marked the first time in race history that the race began outside of Italy. The small country only hosted the stage's start as the stage concluded in Perugia.

To transfer from Sicily to Milan, a plane flight was planned during the rest day. This caused some controversy, because not all riders were happy to fly, and some chose to travel by train.

The twentieth stage was originally planned to end in Solda after 190 km, but was shortened to end on the Passo dello Stelvio because of the risk of an avalanche.

Adorni took the lead in stage six, but lost it in stage eight because of a break-away that took 15 minutes. Adorni re-took the lead in an individual time trial, and increased his lead to a margin that had not been seen since Fausto Coppi won in 1949. There were no time bonuses in 1965.