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 O2 was 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-1 from 1986 to 2016 and further to 124 Mg N year-1 in 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.