Nitrogen (N) is an essential nutrient limiting life, and its biochemical cycling and distribution in rivers have been markedly affected by river engineering construction and operation. Here, we comprehensively analyzed the spatiotemporal variations and driving environmental factors of N distributions based on the long-term observations (from 2004 to 2016) of seven stations in the Three Gorges Reservoir (TGR). In the study period, the overall water quality status of the river reach improved, whereas N pollution was severe and tended to be aggravated after the TGR impoundment. The anti-seasonal reservoir operation strongly affected the variations in N forms. The total nitrogen (TN) concentration in the mainstream of the Yangtze River continuously increased, although it was still lower than that in the incoming tributaries (Wu and Jialing rivers). Further analysis showed that this increase occurred probably because of external inputs, including the upstream (76%), non-point (22%), and point source pollution inputs (2%). Besides, different N forms showed signi cant seasonal variations; among them, the TN and nitrate nitrogen concentrations were the lowest in the impoundment season (October-February), and the ammonia nitrogen concentrations were the highest in the sluicing season (March-May). These parameters varied likely because of internal N transformation. Redundancy analysis revealed that the water level regulated by the anti-seasonal operation was the largest contributor. Our ndings could provide a basis for managing and predicting the water quality in the Yangtze River.