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 mu mol 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.