Nitrate (NO3−) pollution poses a global aquatic threat, promoting eutrophication and endangering human health. Large floods can swiftly drive significant nitrogen load into rivers and downstream water bodies, nevertheless, precisely quantifying NO3− sources in watersheds with complex land use patterns remains challenging. In June 2020, heavy and persistent rainfall triggered severe floods across southeastern China, causing Lake Qiandao, the largest reservoir in this region, to reach its highest recorded level. This study investigated NO3− sources and transformations within the Xin'an River-Lake Qiandao system, particularly focusing on the impact of the catastrophic summer flooding event. Through extensive sampling, we identified a distinct nitrogen concentration gradient: from the pristine Xin'an River source, characterized by 95 % forest cover and consistently low nitrogen levels for the initial 125 km, to a gradual rise as the river traversed human-impacted regions, culminating in peak concentrations within residential and agricultural areas. Isotopic analysis identified a general upward trend in nitrate stable isotope (δ15N–NO3–) associated with the expansion of agricultural and urban lands. Mean values of δ15N–NO3– gradually increased from headwaters and forest-dominant catchments (+2.88 ‰) to agricultural regions (+6.64 ‰) and residential areas (+7.10 ‰). A Bayesian modeling identified that during the flood season, soil erosion and chemical fertilizers were the primary contributors, collectively accounting for 74 % of NO3− sources in the Xin'an River. However, accumulated sewage in tributaries and the overflow of sewage from treatment plants, exacerbated by the flood, significantly impacted Lake Qiandao, particularly in densely populated urban areas, contributing 53 % of the total NO3− input. Additionally, NO3− levels and isotopic values in Lake Qiandao were influenced by a mixture of sources and nitrogen cycling processes, including nitrification and algae assimilation following the flood event. In contrast, during baseflow conditions, the contribution of domestic sewage and livestock wastewaters increased to 41 % in the upper river, while Lake Qiandao remained affected by non-point source pollution, with soil erosion and chemical fertilizers contributing 58 % to the total nitrogen pollution. This study sheds light on the complex dynamics of NO3− dynamics within river–reservoir systems, particularly in the context of extreme flooding, emphasizing the critical need for comprehensive water quality management strategies along the entire watershed.