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Gelsemium is a small genus of flowering plants from the family Loganiaceae comprising five species, three of which, Gelsemium sempervirens (L.) J. St.‐Hil., G. elegans Benth and G. rankinii Small, are particularly popular. Compared with other alkaloids from G. elegans, gelsemine, gelsevirine and koumine exhibit equally potent anxiolytic effects and low toxicity. Although the pharmacological activities and metabolism of koumine and gelsemine have been reported in previous studies, the species differences of gelsevirine metabolism have not been well studied. In this study, the metabolism of gelsevirine was investigated by using liver microsomes of humans, pigs, goats and rats by means of HPLC‐QqTOF/MS. The results indicated that the metabolism of gelsevirine in liver microsomes had qualitative and quantitative species differences. Based on the results, the possible metabolic pathways of gelsevirine in liver microsomes were proposed. Investigation of the metabolism of gelsevirine will provide a basis for further studies of the in vivo metabolism of this drug., The metabolism of gelsevirine was investigated using liver microsomes of human, pig, sheep and rat using a HPLC/QqTOF‐MS method. The results indicated that the metabolism of gelsevirine in liver microsomes almost had a certain extent qualitative and quantitative species‐difference. Based on these results, the possible metabolic pathways of gelsevirine in liver microsomes were proposed. The investigation on the metabolism of gelsevirine will facilitate the development and utilization of G. elegans in clinic and its safety evaluation.