Chiral triazole fungicides have played a significant role in plant pathogen control. Although their enantiomers often exhibit different bioactivity, the mechanism of the stereoselectivity has not been well studied. The stereoselective bioactivity and mechanisms of prothioconazole and its chiral metabolite against plant pathogenic fungi were investigated. The results indicated that the metabolite exerted more fungicidal activities than the activities of the parent compound. R-Prothioconazole and R-prothioconazole-desthio were 6-262 and 19-954 times more potent against pathogenic fungi than the S-enantiomers, respectively. The R-enantiomers were more effective than in inhibiting the biosynthesis of ergosterol and deoxynivalenol the S-enantiomer. Homology modeling and molecular docking suggested that the R-enantiomers of prothioconazole and prothioconazole-desthio possessed better binding modes than S-enantiomers to CYP51B. Moreover, exposure to prothioconazole and its metabolite enantiomers significantly changed the transcription levels of the CYP51 (CYP 51A, CYP51B, CYP 51C) and Tri (Tri5, Tri6, Tri12) genes. The results showed that application of the R-prothioconazole could require a smaller application amount to eliminate the carcinogenic mycotoxins and any environmental risks.,Copyright © 2019 Elsevier Inc. All rights reserved.