In the vast oligotrophic ocean, bioavailable nitrogen has long been limited by scarcity. Nitrogen-fixing organisms input new bioavailable nitrogen into oligotrophic marine ecosystems via biological nitrogen fixation, significantly regulating the regional nitrogen budget balance and primary productivity level. Mesoscale eddies are widespread in oligotrophic ocean, and the vertical mixing and physical disturbance they induce substantially affect nutrient supply and phytoplankton community structure in these regions. Although certain understandings have been gained regarding the biological nitrogen fixation contribution of the unicellular diazotroph cyanobacterium UCYN-B in oligotrophic waters, its response to eddy-driven environmental changes remains poorly understood. In this study, flow cytometry and high-performance liquid chromatography were employed to investigate the distribution characteristics of the N₂-fixing microorganism UCYN-B and non-N₂-fixing picophytoplankton communities within a cyclonic eddy in the Northwestern Pacific Subtropical Gyre. Significant differences in spatial distribution were detected: the abundance of UCYN-B decreased sharply from the eddy outside to the eddy core, while the abundance of other phytoplankton groups exhibited relatively minor variations across different regions of the eddy, maintaining stable biomass. This decoupling indicates that UCYN-B is more sensitive to eddy-induced environmental changes than other phytoplankton, which may be related to its specific ecological niche. We propose that the inhibition of UCYN-B in the eddy core may be associated with changes in nutrient conditions, alterations in temperature and light conditions, and competitive interactions under enhanced mixing. These factors collectively put this nitrogen-fixing group at a disadvantage relative to non-nitrogen-fixing picophytoplankton in the core of cyclonic eddies. Given that the frequency and intensity of mesoscale eddies are projected to increase under future climate scenarios, this differential response of microbial functional groups to mesoscale eddies may have a significant impact on biogeochemical cycles in oligotrophic waters.