In this study, based on molecular technique, 454-pyrosequencing of nifH gene, we investigated the community structure of diazotrophs in the OMZ of Costa Rica upwelling Dome in Eastern Tropical North Pacific Ocean. By comparing the diazotroph communities in different layers of the water columns in the OMZ, unique and more diverse diazotroph community was found in the core of OMZ, which was predominated with methanotroph-like diazotrophs, suggesting the potential coupling of nitrogen cycle and methane assimilation. Besides that, some OTUs reveled in this study were also detected to express the nifH gene in other OMZs, which indicated that the unique hydrographic conditions in the OMZs may shape the community structure of diazotrophs and thus further affect the N cycle.