Springs are an important source of drinking water supply in mountainous karst areas of SW China. However, the quality of many spring waters has deteriorated greatly in recent years, which leads to a significant problem of drinking water scarcity. In this study, hydrochemistry and stable sulfur and oxygen isotopic compositions of SO42- (δ34S and δ18OSO4) of 38 representative samples of waters (incl. spring water, surface water, rainwater, and sewage) from the Hongjiadu Basin, Guizhou province, SW China, were investigated in order to identify the sources of contaminates in spring waters and trace the processes affecting the karst groundwater quality. Approximately 28% of the total investigated springs has been suffered from serious contamination and the concentrations of NO3-, SO42-, and total iron (TFe) in many spring waters have exceeded the standards for drinking water. The springs that have NO3- concentrations of > 30 mg/L are concentrated in residential and agricultural areas, suggesting that NO3- in spring water are mainly derived from chemical fertilizers, manure, and sewage. δ34S and δ18OSO4 data indicate that SO42- in spring water mainly originates from sulfide oxidation, acid rain, and sewage. Furthermore, the high δ34S and δ18OSO4 values of SO42- in some spring waters may be related to the occurrence of bacterial sulfate reduction. Some springs that are discharged from abandoned coal mines have SO42- concentrations of > 250 mg/L, demonstrating that mining activities have accelerated the deterioration of spring water quality. Also, springs with TFe concentrations of > 0.3 mg/L are discharged from coal-bearing strata, revealing that iron in spring waters is mainly derived from the oxidation of pyrite. Our results show that the karst spring waters are highly vulnerable to anthropogenic contaminations and human activities, such as agricultural fertilizing and sewage and waste disposal as well as mining activities, which exert a great impact on the quality of groundwater in karst areas.