In the present study, we report N2 fixation rate (15N isotope tracer assay) and the diazotroph community structure (using the molecular method) in the western tropical North Pacific Ocean (WTNP) (13–20°N, 120–160°E). Our independent evidence on the basis of both in situ N2 fixation activity and diazotroph community structure showed the dominance of unicellular N2 fixation over majority of the WTNP surface waters during the sampling periods. Moreover, a shift in the diazotrophic composition from unicellular cyanobacteria group B-dominated to Trichodesmium spp.-dominated toward the western boundary current (Kuroshio) was also observed in 2013. We hypothesize that nutrient availability may have played a major role in regulating the biogeography of N2 fixation. In surface waters, volumetric N2 fixation rate (calculated by nitrogen) ranged between 0.6 and 2.6 nmol/(L·d) and averaged (1.2±0.5) nmol/(L·d), with <0 μm size fraction contributed predominantly (88%±6%) to the total rate between 135°E and 160°E. Depth-integrated N2 fixation rate over the upper 200 m ranged between 150 μmol/(m2·d) and 480 μmol/(m2·d) (average (225±105) μmol/(m2·d). N2 fixation can account for 6.2%±3.7% of the depth-integrated primary production, suggesting that N2 fixation is a significant N source sustaining new and export production in the WTNP. The role of N2 fixation in biogeochemical cycling in this climate change-vulnerable region calls for further investigations.