Abstract: Laboratory experiments and point monitoring of reservoir sediments have proven that stable sulfate reduction (SSR) can lower the concentrations of toxic metals and sulfate in acidic groundwater for a long time. Here, we hypothesize that SSR occurred during in situ leaching after uranium mining, which can impact the fate of acid groundwater in an entire region. To test this, we applied a sulfur isotope fractionation method to analyze the mechanism for natural attenuation of contaminated groundwater produced by acid in situ leaching of uranium (Xinjiang, China). The results showed that δ34S increased over time after the cessation of uranium mining, and natural attenuation caused considerable, area-scale immobilization of sulfur corresponding to retention levels of 5.3%–48.3% while simultaneously decreasing the concentration of uranium. Isotopic evidence for SSR in the area, together with evidence for changes of pollutant concentrations, suggest that area-scale SSR is most likely also important at other acid mining sites for uranium, where retention of acid groundwater may be strengthened through natural attenuation. To recapitulate, the sulfur isotope fractionation method constitutes a relatively accurate tool for quantification of spatiotemporal trends for groundwater during migration and transformation resulting from acid in situ leaching of uranium in northern China.