Subtropical-subpolar transition water is a potential domain for N-2 fixation, but the understanding of N-2 fixation in such waters remains incomplete. We simultaneously examined the N-2 fixation activity and community structures of diazotrophs and all prokaryotes from the surface to just above the seafloor off Patagonia in the transitional region of the eastern South Pacific Ocean. N-2 fixation activity was not detected in the surface waters, but was observed sporadically and only in subpolar bathypelagic waters (>1,000 m) at very low rates (0.02-0.06 nmol N L-1 d(-1)). By contrast, the nifH gene, a key gene involved in N-2 fixation, was detected widely from the surface to the bottom waters. The majority of diazotrophs were classified as non-cyanobacterial diazotrophs (NCDs), and the nifH amino acid sequences of major diazotrophs were similar to sequences detected in the Southern Ocean, the aphotic zone and sediment of other oceans, and estuarine waters, suggesting that the NCDs are distributed across diverse marine environments. The overall prokaryotic communities were generally similar to those in other open ocean regions at the phylum level (class level for Proteobacteria) and differed among water depths. Diazotrophs, in contrast, showed vertical and horizontal heterogeneity below the euphotic zone and little association with water depth, indicating a lack of cohesion within the community, which may characterize diazotroph community in the transitional surface water and aphotic zones. Elucidating this community heterogeneity may provide pivotal information about N-2 fixation in these waters.