The El Niño-Southern Oscillation (ENSO) is a natural climate phenomenon that alters the biogeochemical and physical dynamics of the Eastern Tropical Pacific Ocean. Its two phases, El Niño and La Niña, are characterized by decreased and increased coastal upwelling, respectively, which have cascading effects on primary productivity, organic matter supply, and ocean-atmosphere interactions. The Eastern Tropical South Pacific (ETSP) oxygen minimum zone (OMZ) is a source of nitrous oxide (N2O), a potent greenhouse gas, to the atmosphere. While nitrogen cycling in the ETSP OMZ has been shown to be sensitive to ENSO, we present the first study to directly compare N2O distributions during both ENSO phases using N2O isotopocule analyses. Our data show that during La Niña, N2O accumulation increased six-fold in the upper 100 m of the water column, and N2O fluxes to the atmosphere increased up to 100-fold. N2O isotopocule data demonstrated substantial increases in δ18O up to 60.5‰ and decreases in δ15Nβ down to -10.3‰, signaling a shift in N2O cycling during La Niña in the oxycline compared to El Niño. N2O production via the hybrid pathway and incomplete denitrification with overprinting of N2O consumption are likely co-occurring to maintain the high site preference (SP) values (17‰ – 26.7‰), corroborating previous hypotheses. Ultimately, our results illustrate a strong connection between upwelling intensity, biogeochemistry, and N2O flux to the atmosphere, and highlight the importance of repeat measurements in the same region to constrain N2O interannual variability and cycling dynamics under different climate scenarios.