Abstract: Bromuconazole is a potent triazole fungicide and consists of two pairs of enantiomers trans(2R, 4R)-, trans(2S, 4S)-configurations and cis(2R, 4S), cis(2S, 4R)-configurations. The degradation of the enantiomers of (2RS, 4RS)- and (2RS, 4SR)-bromuconazole by phase I metabolism was investigated using rat, mouse, rabbit, dog and human liver microsomes. Bromuconazole isomers were quantified using HPLC-MS/MS (ESI⁺) after the separation on a combination of a reversed phase and a chiral analytical column. Moreover, the absolute configuration of four enantiomers had been determined based on the comparisons of the vibrational circular dichroism experimental spectra with the theoretical curve obtained by density functional theory calculations. The degradation of all four isomers followed first-order kinetics and the (2R, 4S)-bromuconazole displayed a significantly longer half-life during microsomal incubation than that of other antipodes. Moreover the metabolic rates of four isomers in human and mouse were much slower than those in rat, rabbit and dog, indicating species-specific differences in the metabolism of bromuconazole. The V_max values for (2S, 4R)-bromuconazole were obviously higher than those of its antipode in all liver microsomes studied. The V_max values for (2R, 4R)-bromuconazole were significantly higher than those for (2S, 4S)-bromuconazole in all liver microsomes studied except human.