[3] I-64 departed Ryojun, Manchukuo, on 27 September 1934 in company with I-61, I-62, and the submarines I-56, I-57, I-58, I-65, I-66, and I-67 to conduct a training cruise in the Qingdao area off China.

[2] While en route, the entire squadron was diverted to Samah on Hainan Island in China.

[2] As a unit of Patrol Group "B," I-64 was among submarines tasked with attacking Allied shipping in the Indian Ocean west of the 106th meridian east, operating from a new base at newly captured Penang in Japanese-occupied British Malaya.

[2] At 16:30 local time on 22 January 1942 while in the Indian Ocean 550 nautical miles (1,020 km; 630 mi) west of Sibolga, Sumatra, she fired two torpedoes at the Dutch 4,482-gross register ton Koninklijke Paketvaart-Maatschappij merchant ship Van Overstraten, which was on a voyage from Bombay, India, to Oosthaven, Sumatra.

[2] She scored a number of hits, slowing Van Overstraten and killing four members of her crew.

[2] At 05:47 GMT on 28 January 1942, I-64 surfaced in the Palk Strait north of Ceylon and opened fire with her deck gun on the 391-gross register ton British Inland Water Transport paddle steamer Idar, which was steaming from Madras to Cochin, India.

[2] Ten minutes later, Florence Luckenbach′s entire crew of 38 abandoned ship in her single surviving lifeboat.

[2] I-64 was in the Bay of Bengal 50 nautical miles (93 km; 58 mi) south of Madras on 31 January 1942 when she torpedoed the British-Indian 4,215-gross register ton cargo steamer Jalapalaka — steaming in ballast from Bombay to Rangoon — at 13:00 GMT.

[2] On 6 March 1942, I-64 set out from Penang to begin her third war patrol, again targeting Allied shipping in the Indian Ocean.

[2] She was in the Indian Ocean off India′s Coromandel Coast 150 nautical miles (280 km; 170 mi) northeast of Madras when she surfaced at around 12:10 GMT and opened fire with her deck gun on the Norwegian 1,513-gross register ton armed cargo steamer Mabella, which was on a voyage in ballast from Colombo, Ceylon, to Calcutta, India.

[2] On 16 May 1942 she departed Sasebo bound for Kwajalein, deploying to support Operation MI,[2] the invasion of Midway Atoll planned for early June 1942.

At 18:03 on 17 May 1942, the United States Navy submarine USS Triton (SS-201) sighted I-64 on the surface in the Pacific Ocean 250 nautical miles (460 km; 290 mi) south-southeast of Cape Ashizuri, Shikoku, Japan.