First commissioned in 1925, she served in the waters of Japan and Chōsen prior to World War II.

During World War II, she operated in the Central Pacific, supported the Japanese invasion of Rabaul, and took part in the Aleutian Islands campaign, then in late 1942 was relegated to a role as a training ship.

For surface running, the submarines were powered by two 1,200-brake-horsepower (895 kW) Vickers diesel engines, each driving one propeller shaft.

[2] On 1 June 1925, she was transferred to the Sasebo Naval District and reassigned to Submarine Division 24, in which she remained until 1939.

[2] On 1 March 1926, Ro-64 and the submarines Ro-57, Ro-58, Ro-59, Ro-60, Ro-61, Ro-62, Ro-63, and Ro-68 departed Sasebo, Japan, bound for Okinawa, which they reached the same day.

[2][4][5][6][7][8][9][10][11] The nine submarines got underway from Okinawa on 30 March 1926 for a training cruise in Chinese waters off Shanghai and Amoy which concluded with their arrival at Mako in the Pescadores Islands on 5 April 1926.

[2] She helped Royal Thai Navy submarine crews perform dive training sometime after September 1937.

[3] Her commanding officer initially intended to send a landing party ashore to destroy the surviving facilities, but decided against the landing at 22:40 Japan Standard Time (JST) out of a concern that defenders ashore had sighted Ro-64 and because of a relatively high sea state.

[3] He decided instead to shell Howland with Ro-64′s 76.2-millimeter (3 in) deck gun, and at 02:00 JST on 11 December 1941 Ro-64 began her bombardment, firing at the island′s wireless and weather station, barracks, and lighthouse.

[3] She then patrolled south of Cape St. George on New Ireland in support of Japanese forces landing at Rabaul before she returned to Truk on 29 January 1942.

[3] During their voyage, however, Ro-63′s horizontal rudder failed on 27 February 1942,[3][13] and Ro-64 accompanied her as she proceeded to Bikini Atoll for repairs.

At 16:00 on 24 July 1942, Ro-63, Ro-64, and Ro-68 departed Yokosuka, Japan, bound for Paramushiro in the northern Kurile Islands,[3] but an outbreak of food poisoning among her crew forced Ro-64 to turn back.

[13][14] On 7 August 1942, an American task force bombarded Kiska while I-6, Ro-61, Ro-64, and Ro-68 were anchored in the harbor, and they crash-dived to avoid damage.

[3] On 28 August 1942, a Kiska-based Aichi E13A1 (Allied reporting name "Jake") reconnaissance floatplane sighted the U.S. Navy seaplane tender USS Casco (AVP-12) — which the plane′s crew mistakenly identified as a light cruiser — and a destroyer in Nazan Bay on the coast of Atka.

[2] At 14:28 JST on 12 April 1945, Ro-64 was submerged in Hiroshima Bay during a training cruise when she detonated a magnetic mine laid by an American aircraft.

[3] She sank quickly at 34°14′N 132°16′E / 34.233°N 132.267°E / 34.233; 132.267 (Ro-64)[2] with the loss of all 81 men on board — her crew of 50, the embarked commander of Submarine Division 33, and 30 trainees.