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Many estuaries experience eutrophication, deoxygenation and warming, with potential impacts on greenhouse gas emissions. However, the response of N₂O production to these changes is poorly constrained. Here we applied nitrogen isotope tracer incubations to measure N₂O production under experimentally manipulated changes in oxygen and temperature in the Chesapeake Bay—the largest estuary in the United States. N₂O production more than doubled from nitrification and increased exponentially from denitrification when O₂was decreased from >20 to <5 micromolar. Raising temperature from 15° to 35°C increased N₂O production 2- to 10-fold. Developing a biogeochemical model by incorporating these responses, N₂O emissions from the Chesapeake Bay were estimated to decrease from 157 to 140 Mg N year⁻¹from 1986 to 2016 and further to 124 Mg N year⁻¹in 2050. Although deoxygenation and warming stimulate N₂O production, the modeled decrease in N₂O emissions, attributed to decreased nutrient inputs, indicates the importance of nutrient management in curbing greenhouse gas emissions, potentially mitigating climate change.