Nitrous oxide (N2O) emissions from lakes exhibit significant spatiotemporal heterogeneity, and quantitative identification of the different N2O production processes is greatly limited, causing the role of nitrification to be undervalued or ignored in models of a lake's N2O emissions. Here, the contributions of nitrification and denitrification to N2O production were quantitatively assessed in the eutrophic Lake Taihu using molecular biology and isotope mapping techniques. The N2O fluxes ranged from -41.48 to 28.84 μmol m-2 d-1 in the lake, with lower N2O concentrations being observed in spring and summer and significantly higher N2O emissions being observed in autumn and winter. The 15N site preference and relevant isotopic evidence demonstrated that denitrification contributed approximately 90% of the lake's gross N2O production during summer and autumn, 27-83% of which was simultaneously eliminated via N2O reduction. Surprisingly, nitrification seemed to act as a key process promoting N2O production and contributing to the lake as a source of N2O emissions. A combination of N2O isotopocule-based approaches and molecular techniques can be used to determine the precise characteristics of microbial N2O production and consumption in eutrophic lakes. The results of this study provide a basis for accurately assessing N2O emissions from lakes at the regional and global scales.