Prothioconazole (PTA) is a novel, broad-spectrum, chiral triazole fungicide that is mainly used to prevent and control the disease of cereal crops. However, the adverse effects of PTA and its major metabolite on nontarget organisms have aroused wide concern. In the present work, the acute toxic of the metabolite prothioconazole-desthio (PTA-desthio), with an LC50 of 1.31 mg L-1, was 3.5-fold more toxic than the parent compound, indicating that the metabolism of PTA in zebrafish was toxic. The stereoselective uptake and metabolism of PTA and PTA-desthio in zebrafish was firstly investigated using LC-MS/MS. Remarkable enantioselectivity was observed: S-PTA and S-PTA-desthio were preferentially uptake with the uptake rate constants of 8.22 and 8.15 d-1 at exposure concentration of 0.5 mg L-1, respectively, and the R-PTA-desthio were preferentially metabolized. PTA-desthio was rapidly formed during the uptake processes. The antioxidant enzyme activities in the zebrafish changed significantly, and these effects were reversible. A metabolic pathway including 13 phase I metabolites and 2 phase II metabolites was firstly proposed. A glucuronic acid conjugate and sulfate conjugate were observed in zebrafish. The results of this work provide information that highlights and can help mitigate the potential toxicity of PTA to the ecological environment and humans health.