In the modern ocean, N₂ fixation serves as a major source of bioavailable nitrogen to the ocean and support biologic productivity in nitrate-limited oligotrophic subtropical gyres. Yet variations in N₂ fixation across these regions remain poorly constrained, particularly during glacial–interglacial cycle characterized by contrasting climatic regimes and major perturbations to the marine nitrogen reservoir. Here we present a new foraminifera-bound δ¹⁵N (FB-δ¹⁵N) record from the North Pacific Subtropical Gyre (NPSG) spanning the past 24 ka. Our FB-δ¹⁵N record exhibits a glacial–interglacial amplitude of ~1.5‰, reaching relatively elevated δ¹⁵N values during the early Holocene. Our data reveal comparable intensities of N₂ fixation during the Last Glacial Maximum (LGM) and the late Holocene, with a gradual decline from the LGM to the early Holocene. We suggest that enhanced aeolian iron supply during the LGM elevated surface-water Fe/N ratios, thereby stimulating diazotroph proliferation, whereas reduced aeolian iron flux during deglaciation attenuated this stimulation on regional N₂ fixation. The aeolian iron input stimulated N₂ fixation, thereby alleviating glacial nitrogen limitation and enhancing regional biological productivity during LGM. Our records provide direct evidence for the regulatory role of aeolian iron on N₂ fixation, and demonstrate that N₂ fixation in the NPSG during the LGM was comparable to that of the Holocene, a finding that contrasts markedly with predicted LGM declines of N₂ fixation. Our findings highlight the regional heterogeneity of oceanic N₂ fixation variability and its driving mechanisms on glacial–interglacial timescales, such spatial disparities must be considered in both past reconstructions and future projections of the oceanic nitrogen budget.