In the present study, metabolites of T-2 toxin in in vivo and in vitro systems of Wistar rats were identified and elucidated by ultra-performance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS). Expected and unexpected metabolites were detected by MetabolynxXS software which could automatically compare MSE data from the sample and control. A total of 19 metabolites of T-2 toxin were identified in this research, 9 of them being novel, which were 15-deacentyl-T-2, 3'-OH-15-deacentyl-T-2, 3',7-dihydroxy-T-2, isomer of 3',7-dihydroxy-T-2, 7-OH-HT-2, isomer of 7-OH-HT-2, De-epoxy-3',7-dihydroxy-HT-2, 9-OH-T-2 and 3',9-dihydroxy-T-2. The results showed that the main metabolic pathways of T-2 toxin were hydrolysis, hydroxylation and de-epoxidation. In addition, the results also revealed one novel metabolic pathways of T-2 toxin, hydroxylation at C-9 position which was demonstrated by the metabolites of 9-OH-T-2 and 3',9-dihydroxy-T-2. In addition, hydroxylation at C-9 of T-2 toxin was also generated in in vitro of liver systems. Interestingly, several metabolites of hydroxylation at C-7 of T-2 toxin were also detected in in vivo of male Wistar rats, while they were not found in in vivo of female rats and in in vitro systems of Wistar rats.