A unicellular diazotrophic cyanobacterium strain of Group C, designated TW3, was isolated from the oligotrophic Kuroshio Current of the western Pacific Ocean. To our knowledge, this represents the first successful laboratory culture of a Group C unicellular diazotroph from oceanic water. TW3 cells are green rods, 2.5–3.0 µm in width and 4.0–6.0 µm in length. Phylogenetic analyses of both 16S rRNA and nifH gene fragments indicated that the TW3 sequences were over 98% identical to those of the previously isolated Cyanothece sp. ATCC51142 and Gloeocapsa sp., suggesting that TW3 is a member of the Group C unicellular diazotrophs. In addition, both TW3 and Cyanothece sp. ATCC51142 share morphological characteristics; both strains are sheathless and rod-shaped, display binary fission in a single plane, and possess dispersed thylakoids. TW3 grows aerobically in nitrogen-deficient artificial seawater, and exhibited the highest observed growth rate of 0.035 h−1 when cultured at 30°C and 140 µmol m−2 s−1 of light intensity. The nitrogen fixation rate, when grown optimally using a 12 h/12 h light–dark cycle, was 7.31 × 10−15 mol N cell−1 day−1. Immunocytochemical staining using Trichodesmium sp. NIBB1067 nitrogenase antiserum revealed the existence of diazotrophic cells sharing morphological characteristics of TW3 in the Kuroshio water from which TW3 was isolated.