Prior to WWII HMS REGULUS was based with the 4^th Submarine Flotilla at Hong Kong. After the outbreak of WWII she carried out four War Patrols from Hong Kong. The first was in the South China Sea, the second and third were in Japanese waters and the fourth was in Russian waters off Vladivostock.

Following a Refit at Singapore HMS REGULUS visited Saigon in French Indo-China and, after returning to Hong Kong, the submarine was sent to the Mediterranean to join the 1^st Submarine Flotilla based at Alexandria in Egypt.

In the Mediterranean HMS REGULUS conducted two War Patrols returning each time to Alexandria. HMS REGULUS (Lt. Cdr. Frederick Basil Currie, RN) left Alexandria for a third Mediterranean War Patrol in the Southern Adriatic on 23 November 1940. She was was lost on about 26 November 1940 in the Strait of Otranto.

The Italians claimed to have sunk a British submarine by bombing on 26 November 1940 off Cyrenaica but only OSIRIS and OTUS were in that area at the time, and neither was attacked.

The Italians also claimed on 10 January 1941 that REGULUS had been sunk by torpedo bomber attack on an unspecified date. This claim was made after the Admiralty announcement of her loss and can probably be discounted.

The Adriatic was reported to be mined and the Italians had broadcast warnings of floating mines in the Strait of Otranto, so it is probable that REGULUS struck one. The submarine was declared lost on failure to return from patrol on 6 December 1940.