The biogeochemical cycling on the Qinghai–Xizang Plateau is sensitive to climate change. Yet the response of fluvial nitrate (NO3−) dynamics to climatic conditions on the plateau is poorly understood. Here, natural abundance isotopes were synthesized and15N pairing experiments were carried out to systematically clarify the drivers of NO3−-N levels in an alpine river on the southeastern Qinghai–Xizang Plateau in winter. The natural abundance isotopes (δ15N/δ18ONO3) of the river waters suggested that soil organic nitrogen (SON) was the primary source of riverine NO3−-N (96.5% ± 2.2%), and substantial biological NO3− removal occurred in the catchment. The15N pairing techniques quantified that the removal of NO3− in soils and river sediments, i.e., dissimilatory NO3− reduction to ammonium (DNRA) denitrification, and anammox, was prevalent, which outcompeted nitrification. As SON dominated the NO3− sources, we focused on the drivers of NO3− production-related processes in the soils. The denitrification rates in the soils were largely controlled by moisture. The weak remineralization of SON in winter led to high C/N ratios, which favored DNRA. Anammox could be explained by the competition of its substrate (nitrite [NO2−]) with other processes. Low temperatures in winter limited nitrification. The hydrological isotopes (i.e., δ2H/δ18OH2O) showed that NO3− in the soils was supply limited. Higher runoff due to weaker evaporation and more glacial meltwater recharging exerted dilution effects on the NO3−-N levels in the river. This study systematically unraveled the processes regulating the NO3−-N levels in an alpine river in the cold season, providing a window for understanding fluvial NO3−-N dynamics on the plateau.