Spearfish conducted sea trials off New London, Connecticut, and then held her shakedown cruise in the Guantanamo Bay area from 21 August to 3 October.

Spearfish operated between Hawaii and the West Coast until 23 October 1941, when she departed Pearl Harbor and headed for Manila.

This mission took her into the South China Sea, near Saigon and Cam Ranh Bay, French Indochina, and off Tarakan and Balikpapan, Borneo.

On 2 March, she put into Tjilatjap, Java, and took on board 12 members of the staff of the commander of the submarines of the Asiatic Fleet, for transportation to Australia.

On the night of 3 May, the submarine slipped into Manila Bay and picked up 14 nurses, and several staff officers from Corregidor.

From 26 June to 17 August, she scouted the South China Sea for enemy shipping, and from 8 September to 11 November, searched the west coast of Luzon, where she damaged two freighters.

Spearfish sailed from Brisbane on 2 December 1942 and patrolled in the New Britain-New Ireland area for over a month before entering Pearl Harbor on 25 January 1943.

Spearfish sailed on her 11th war patrol from Pearl Harbor, 31 March, for the East China Sea and the area north of Nansei Shoto.

She was on the surface 23 nautical miles (43 km; 26 mi) east-southeast of Iwo Jima at 24°40′N 141°39′E / 24.667°N 141.650°E / 24.667; 141.650 at 17:15 on 28 November 1944 when a United States Army Air Forces B-24 Liberator bomber attacked her with rockets and strafed her.

[6] On 19 December 1944, Spearfish rescued seven survivors (CPT Linden O. Bricker, 2LT Kenneth R. Chidester, 2LT Jay L. Meikle, 2LT Clifford B. Smith, SGT Edmund G. Smith, CPL Stephen J. Darienzo, and SSG Richard J. Grinstead) from a ditched B-29 Superfortress, Z-1 Pee Wee.

This marked the first submarine rescue of downed B-29 airmen in the Pacific theater of operations during World War II.

Cole, commanding officer of the Spearfish, noted in the ship's log at Tanapag Harbor, Saipan, on 13 January that he returned the seven airmen to their quarters under jubilant escort from their squadron.

She was stricken from the Naval Vessel Register on 19 July 1946, sold to the Lerner Company of Oakland, California, and scrapped in October 1947.