Biological nitrogen (N₂) fixation is an important source of bioavailable nitrogen to the oligotrophic ocean, supporting primary production and thus influencing the global carbon cycle. Despite its importance, a systematic understanding of diazotroph biogeography and its controls within and between the ocean basins is still lacking. Here we investigate the abundance and activity of diazotrophic microorganisms across the subtropical western North Pacific, spanning from the western boundary Kuroshio Current to the North Pacific Subtropical Gyre (NPSG). We further carry out nutrient-addition incubation experiments to assess which nutrients are limiting N₂ fixation in the study region. The results demonstrate a switch of both N₂ fixation and its limiting nutrient across the subtropical western North Pacific. High N₂ fixation rates occur mainly between 15-25°N (center of the gyre) in summer and decrease significantly toward the southern and northern edges of the NPSG. Both functional gene sequencing and qPCR of the nifH gene indicate that UCYN-B is the most dominant diazotroph in the NPSG. The biogeography of N₂ fixation rate at the basin scale is regulated by the supply ratio of iron to nitrogen in the upper ocean, and the spatial and seasonal distribution of diazotrophs community structure is mainly regulated by temperature and nutrients. In addition, the niches characterized mainly by dissolved iron, dissolved inorganic phosphorus, and temperature of the three major N2-fixers, i.e., Trichodesmium, UCYN-A and UCYN-B are distinct. This study reveals the spatial and seasonal patterns of biological N₂ fixation in the NPSG, which also provides insight into biogeochemcial cycling of carbon in the NPSG.