Prothioconazole is a widely used chiral triazole fungicide, and its residue pollution has attracted wide attention in recent years. However, little is known about microbial metabolic processes of prothioconazole enantiomers. In this study, a prothioconazole-degrading strain, Sphingomonas sp. AJ-1, was isolated from activated sludge. The optimal temperature and pH for prothioconazole degradation by strain AJ-1 were 30 °C and 6.0, respectively, and the degradation rate of prothioconazole by strain AJ-1 was negatively correlated with the initial concentration. When supplemented with additional carbon source, the degradation rates of 10 mg/L (Rac)-/(S)-/(R)-prothioconazole by strain AJ-1 were 76.0 %, 100.0 % and 64.8 % within 6 d, respectively. The CS bond of prothioconazole was methylated to produce (S)-/(R)-prothioconazole-S-methyl by strain AJ-1, but the degradation rate of prothioconazole by strain AJ-1 with (S)-enantiomer was 2.54-fold of that with (R)-enantiomer. Moreover, the toxicity of (Rac)-prothioconazole-S-methyl was 5.57 times lower than that of (Rac)-prothioconazole to Pseudokirchneriella subcapitata. The results showed that strain AJ-1 had obvious enantioselective metabolism for prothioconazole, and this metabolism was a detoxification process. This study provides new insights into the enantioselective metabolism of the chiral fungicide prothioconazole in microorganisms.