The oxidation of Sb(Ⅲ) occurred rapidly in aerobic and dark groundwater environments, with previous studies suggesting that co-oxidation of Fe(Ⅱ) and Sb(Ⅲ) may be the predominant driving mechanism. However, there is a lack of field evidence confirming environmental isotope fractionation. Therefore, 20 groups of Magunao aquifer (water) samples were collected from the North mine of Xikuangshan antimony mining area in Hunan Province to compare the differences in environmental isotopes (δ⁵⁶ Fe,δ¹³ C, and δ³⁴ S) between high- and low-Sb groundwater and investigate the fractionation process of these isotopes. The results reveal that total Sb(^TSb) concentrations ranged from 5.30 μg/L to 20 700 μg/L, with a mean concentration of 3 660.61 μg/L. Additionally, Sb(V) is found to be the most dominant valence state for Sb in water. The neutral-alkaline and oxygen-enriched conditions in water facilitate the co-oxidation of FeS₂ and Sb₂ S₃, as well as induce fractionation of δ¹⁸ O_SO4, δ³⁴S_SO4 and δ⁵⁶Fe between sediments and groundwater, resulting in the increase of , total Fe (^TFe) and Sb(Ⅴ) contents in high Sb groundwater. Furthermore, microbial activities promote the oxidative decomposition of organic carbon, thereby enhancing the co-oxidation rate of Fe(Ⅱ) and Sb(Ⅲ). This conclusion unveils a novel mechanism for aerobic oxidation of Sb(Ⅲ) in dark groundwater environments while providing a scientific foundation for advancing our understanding of the Sb geochemical cycle and preventing environmental pollution from high Sb groundwater.