Zhijin coal-mining district, located in Midwestern Guizhou Province, has been extensively exploited for several decades. The discharge of acid mine drainage (AMD) has constituted a serious threat to local water environmental quality, which greatly affected the normal use of local people. The Permian limestone aquifer is the essential potable water supply for local people, which covered under the widely distributed coal seams. To investigate the origin of the water, the evolutionary processes, and the sources of dissolved sulfate in the karst waters, the mine water, surface water, and groundwater near the coal mines were sampled for stable isotopes (H, O, and S) and conventional hydrochemical analysis. The results of hydrochemistry and isotopic composition indicate that the regional surface water and partial karst groundwater are obviously affected by coal-mining activities, which is mainly manifested in the increase of water solute concentration and the change of hydrochemical types. The isotopic composition of δ2HH2O and δ18OH2O indicates that the major recharge source of surface water and the groundwater is atmospheric precipitation and that it is influenced obviously by evaporation in the recharge process. The surface water is mainly controlled by the oxidation of pyrite, as well as the dissolution of carbonate rocks, whereas that of natural karst waters is influenced by the dissolution of carbonate rocks. The resulting δ34SSO4 values suggest that the dissolved sulfate source in the surface water is mainly pyrite oxidation but atmospheric precipitation for the karst groundwater. Given the similar chemistry and isotopic composition between surface water and partial groundwater, it is reasonable to assume that most of the dissolved sulfate source in part of the groundwater was derived through the oxidation of pyrite in the coal. Furthermore, the contamination of the surface water and partial groundwater from the coal seam has occurred distinctly in the catchment, which is enriched in SO42- and is mostly depleted δ34S in sulfate.