Karst water resources, which are critical for the support of human societies and ecological systems in many regions worldwide, are extremely sensitive to mining activities. Identification and quantification of stable isotope (δ2HH2O andδ18OH2O) composition for all sources is essential if we are to fully understand the dynamics of these unique systems and propose successful remediation strategies. For these purposes, a stable isotope study was undertaken in two similar watersheds, one impacted by acid mine drainage, and the other not. It was found that the majority of δ2HH2O and δ18OH2O values of acid mine drainage (AMD), AMD-impacted and Main channel mix waters plotted above the local meteoric water line (LMWL), while the non-AMD-impacted water was below the LMWL. The AMD and AMD-impacted water had a similar composition ofδ18OH2O and heavierδ2HH2O than that of the other waters as a result of pyrite oxidation and Fe hydrolysis. The non-AMD-impacted and spring waters were the background waters in the study area. The composition ofδ2HH2O and δ18OH2O for the former was influenced by the re-evaporation and water–rock interaction, and that for the latter was controlled by re-condensation. Along the water flow, the Main channel mix water is recharged by AMD-impacted, non-AMD-impacted and spring waters. The composition ofδ2HH2O andδ18OH2O for the Main channel mix water was coincident with the characteristics of water mixing, supported by three-component mixing modeling of upstream spring, non-AMD-impacted and AMD-impacted waters. The composition of δ2HH2O and δ18OH2O for the Main channel mix water was mainly affected by the AMD-impacted water. These results help elucidate the impact of AMD on δ2HH2O and δ18OH2O compositions for karst waters and demonstrate the utility for impact assessments and remediation planning in these unique systems. •Elucidating δ18OH2O and δ2HH2O dynamics benefits remediation strategies.•Isotopic compositions with and without acid mine drainage were very different.•Isotopic variability was affected by pyrite oxidation and Fe hydrolysis.•The observation was supported by three-component mixing modeling.•Results present models for isotopic fractionation of mine drainage in karst settings.