Abstract: The purpose of current study is to investigate the metabolic profile of a triptolide derivative (5R)-5-hydroxytriptolide in vitro. (5R)-5-Hydroxytriptolide was incubated with the hepatocytes of human, monkey, dog, rat or mouse, respectively. Compared with inactivated hepatocytes, four metabolites were identified in hepatocytes from all five species:oxidative ring-opening metabolite (M1), glutathione-conjugating metabolite (M2), and monooxidative combined with glutathione-conjugating metabolites (M3-1 and M3-2), respectively. In human or rat liver microsomes, seven metabolites of (5R)-5-hydroxytriptolide were found, dehydrogenated metabolite (M4) and monooxidative metabolites (M5-1-M5-6), respectively. Reference standards for the metabolites were obtained either through chemical semisynthesis or biotransformation through rat primary hepatocytes. The structures of five metabolites were confirmed, which were 12,13-epoxy ring-opening metabolite M1, 12-glutathione-conjugating metabolite M2, (16S)-, (2R)- and (19R)-monohydroxylated metabolites M5-1, M5-4, and M5-5, respectively. In vitro activity assay revealed that only (2R)-hydroxylated metabolite exhibited weak immunosuppressive activity with less than one-tenth the activity of its parent drug, and a significant decrease in toxicity was observed. It is suggested that (5R)-5-hydroxytriptolide might undergo metabolic inactivation and detoxification in vivo.