Batatasin III is a biologically active ingredient extracted from Dendrobium scabrilingue, which has been demonstrated to have anticancer activity. To fully understand its action, the present study was performed to investigate the in vitro metabolism of batatasin III using rat and human liver microsomes and hepatocytes. Batatasin III (20 μM) was incubated in the presence of NADPH-supplemented rat and human liver microsomes (0.5mg protein/mL) and hepatocytes (1 million cells/mL) followed by liquid chromatography in combination with hybrid quadrupole Orbitrap tandem mass spectrometric analysis to detect and identify the generated metabolites. The structures of the metabolites were characterized by comparing the accurate masses, elemental compositions as well as indicative fragment ions with those of the parent. A total of 15 metabolites were detected and identified, including 4 phase I and 11 phase II metabolites. Batatasin III is subjected to bioactivation to form reactive quinoid intermediates, which subsequently react with glutathione (GSH) via Michael addition. Glucuronidation and GSH conjugation appear to be the primary elimination pathways in rat hepatocytes, while in human hepatocytes, GSH conjugates are formed to a lesser extent. Phase I metabolic pathways include hydroxylation and demethylation. The present study sheds light on the in vitro metabolic fates of batatasin III, which is indispensable for an understanding of its efficacy and safety.