Denitrification removes ecologically available nitrogen (N) from the biosphere and influences both the pace and magnitude of global climate change. Disagreements exist over the degree to which this microbial process influences N-availability patterns across Earth's ecosystems. correlates positively with δ 18 O-NO 3 − (P ⩽ 0.03) and nirS abundance (P = 0.00002) across sites, revealing the widespread importance of isotopic discrimination by soil denitrifiers. Furthermore, NO 3 − concentrations correlate negatively to nirS (P = 0.002) and δ 15 N-NO 3 − (P = 0.003) across sites. We also observe these spatial relationships in short-term (7-day), in situ soil-incubation experiments; NO 3 − -depletion strongly corresponds with increased nirS, nirS/16 rRNA, and enrichment of heavy NO 3 − isotopes over time. Overall, these findings suggest that microbial denitrification can consume plantavailable NO 3 − to low levels at multiple time scales, contributing to N-limitation patterns across sites, particularly in moist, carbon-rich soils. Furthermore, our study provides a new approach for understanding the relationships between microbial gene abundance and terrestrial ecosystem functioning.