Total nitrogen in Taihu Lake, China has gradually decreased since 2015 while the total phosphorus concentration has exhibited an increasing trend, indicating an asynchronous change. The dominant nitrogen removal process in freshwater ecosystems is denitrification which primarily occurs at the sediment–water interface. In this study, 15N isotope incubation experiments were attempted to analyze the effect of water temperature on denitrification, to construct the regional denitrification Arrhenius equations considering water temperature, and to identify the nitrate source of denitrification in Lake Taihu sediments. The results indicated that the potential N2 production rates and denitrification rates generally decreased in the west to east direction, which was significantly positively correlated with the nitrate concentration of overlying water by Pearson correlation coefficient analysis (P<0.05). In addition, when the water temperature was lower than 30°C, the rates of the potential N2 production and denitrification were higher with an increase in water temperature, but when the water temperature was overhigh, denitrification was inhibited. The ratio of the total denitrification rate of nitrate from the water column in the sediment to the total denitrification rate during the incubation experiment was above 0.5 at each sampling site. This indicated that the denitrification in the Lake Taihu sediment primarily occurred at the expense of nitrate from the water column. Research on denitrification has important implications for improving the water quality of Lake Taihu, and the findings of the study act as a reference for the water environment treatment of other shallow eutrophic lakes in China and globally.