Globally, shallow lakes have suffered from excessive nitrogen (N) loading due to increased human activities in catchments, resulting in water quality degradation and aquatic biodiversity loss. Sediment denitrification, which reduces nitrate (NO3-) to N gaseous products, is the most important mechanism for permanent N removal in freshwater lakes. However, the relative contribution of abiotic and biotic factors to the sediment denitrification is highly variable. Here, we determined the unamended denitrification rate and nitrous oxide (N2O) production rate of 74 sediment samples from 22 eutrophic lakes in the Yangtze River basin. We also quantified the diversity and abundance of denitrifying communities using nirK and nirS genes. The results of variance partitioning analyses showed that water physicochemical properties (e.g., dissolved oxygen) and nutrients (e.g., NO3- concentration) but not denitrifier communities and submerged vegetation were the major factor groups predicting denitrification and N2O production rates. Path analyses further revealed that water physicochemical properties and nutrients could affect denitrification and N2O production rates both directly and indirectly, and the direct effects were considerably higher than the indirect effects mediated through changes in sediment characteristics, denitrifier communities and submerged vegetation. These findings suggest that the dominant N removal process in Yangtze lakes is largely regulated by abiotic factors rather than diversity and abundance of denitrifiers and submerged macrophytes. Additionally, the findings in this study are helpful in developing a targeted strategy to assess and enhance the N removal capability of eutrophic lakes in China.