Nitrous oxide (N 2 O) is atmospheric trace gas that contributes to climate change and affects stratospheric and ground-level ozone concentrations. Ammonia oxidizers and denitrifiers contribute to N 2 O emissions in estuarine waters. However, as an important climate factor, how temperature regulates microbial N 2 O production in estuarine water remains unclear. Here, we have employed stable isotope labeling techniques to demonstrate that the N 2 O production in estuarine waters exhibited differential thermal response patterns between nearshore and offshore regions. The optimal temperatures (T opt ) for N 2 O production rates (N 2 OR) were higher at nearshore than offshore sites. 15 N-labeled nitrite ( 15 NO 2 - ) experiments revealed that at the nearshore sites dominated by ammonia-oxidizing bacteria (AOB), the thermal tolerance of 15 N-N 2 OR increases with increasing salinity, suggesting that N 2 O production by AOB-driven nitrifier denitrification may be co-regulated by temperature and salinity. Metatranscriptomic and metagenomic analyses of enriched water samples revealed that the denitrification pathway of AOB is the primary source of N 2 O, while clade II N 2 O-reducers dominated N 2 O consumption. Temperature regulated the expression patterns of nitrite reductase (nirK) and nitrous oxide reductase (nosZ) genes from different sources, thereby influencing N 2 O emissions in the system. Our findings contribute to understanding the sources of N 2 O in estuarine waters and their response to global warming.