The in vitro metabolism of hirsutine was determined using liver microsomes and human recombinant cytochrome P450 enzymes. Under the current conditions, a total of 14 phase I metabolites were tentatively identified. Ketoconazole showed significant inhibitory effect on the metabolism of hirsutine. Human recombinant cytochrome P450 enzyme analysis revealed that metabolism of hirsutine was mainly catalysed by CYP3A4. Our data revealed that hirsutine was metabolised via mono-oxygenation, di-oxygenation, N-oxygenation, dehydrogenation, demethylation and hydrolysis. In glutathione (GSH)-supplemented liver microsomes, four GSH adducts were identified. Hirsutine underwent facile P450-mediated metabolic activation, forming reactive 3-methyleneindolenine and iminoquinone intermediates. This study provided valuable information on the metabolic fates of hirsutine in liver microsomes, which would aid in understanding the hepatotoxicity caused by hirsutine or hirsutine-containing herb preparation. © 2023 Informa UK Limited, trading as Taylor & Francis Group.