Understanding the heavy metal (HM) contamination in alpine mountain headwaters regions is important to maintaining the ecosystem stability of the basin. A total of 119 water samples and 104 sediment samples were collected along tributaries and the main course of Heihe River. The concentrations of eight heavy metals (As, Cd, Cr, Cu, Mn, Ni, Pb, and Zn) in water and sediment were measured to describe their spatial variability and to assess water quality. To identify the origins and pathways of HMs, anions, cations, and trace elements, as well as δD/δ¹⁸O stable isotopes in water samples were also measured. The results of water quality assessment suggested that tributaries were affected by local mining activity. Factor analysis in sediments showed that all HMs in sediments were inherited from the parent bedrock. Both natural weathering and mining contribute HMs. Cr and Ni were homologous with a source from the weathering of basic gabbro and serpentine at Yushigou. Mn appeared to be influenced more by artificial activities such as agriculture and grazing. Depending on the mining technique involved, two pathways for the release of HMs were distinguished in this area. For open-pit mining, mining promoted the release of HMs primarily via enhanced weathering. For underground mining, HMs might have contributed to greater acid mine discharge at high elevations due to the weak weathering processes. As the elevation decreases, precipitation increases, and a series of complex hydrological factor significantly affect leaching and runoff. The study results can be applied to improve water management efficiency.