The squadron was formed as No 501 (City of Bristol) Squadron as part of the Special Reserve squadrons in June 1929, made up of volunteers and regulars. In 1930 it became "No 501 (County of Gloucester) Squadron". On 1 May 1936 it was transferred to the Auxiliary Air Force.

When war was declared in September 1939, 501 Squadron was based at RAF Filton, near Bristol. On 10 May 1940, the Squadron moved to France where it saw extensive action. After the retreat from France, its battle-hardened pilots were stationed at Gravesend up to 10 September 1940. It subsequently served at RAF Kenley, south London, until 17 December 1940. At Kenley, the squadron was commanded by Harry Hogan and consisted of Hawker Hurricanes.

The squadron included several fighter aces of WWII: Ken Mackenzie,"Ginger" Lacey, Stanislaw Skalski, Robert Dafforn and Kenneth Lee. Ginger Lacey was one of the highest scoring pilots in the Battle of Britain. Robert Dafforn was the tallest man in Fighter Command, and had great difficulty in fitting into his Hurricane.

The squadron included a number of Polish airmen, one of whom, Steve Woltanski, reputedly never spoke English.

The Squadron was disbanded in 1957 along with all the Auxiliary units. However, it was reformed in the Force Protection role in 2001 at RAF Brize Norton. In 2003, its personnel deployed as part of Operation Telic, the liberation of Iraq. The squadron continues to deploy personnel on Force Protection duties in this region.