Abstract: Astrapterocarpan (AP) is a bioactive constituent of Astragali Radix and was selected as a model compound for investigating the in vitro metabolism of pterocarpans in this study. Its in vitro metabolism was conducted by incubation with rat hepatic 9000g supernatant (S9) in the presence of an NADPH-generating system. At first, four compounds were isolated and their structures were elucidated as 6a-hydroxy-AP ( M1 ), astrametabolin Ⅰ M2 , 1a-hydroxy-9, 10-dimethoxy-pterocarp-1(2), 4-diene-3-one, 9-demethyl-AP ( M3 , nissolin) and 4-methoxy-astraisoflavan ( M4 , 7, 2'-dihydroxy-4, 3', 4'-trimethoxy-isoflavan) on the basis of NMR data, respectively. Among them, M1, M2 and M4 were new compounds. Next, the metabolite profile of AP in rat hepatic S9 was obtained via HPLC-DAD-ESI-IT-TOF-MSⁿ, and 40 new metabolites were tentatively identified. These newly identified metabolites included 9 monohydroxylated metabolites, 1 demethylated metabolite, 7 demethylated and monohydroxylated metabolites, 4 dihydroxylated metabolites, 1 hydration metabolite, 1 didemethylated metabolite, 2 glucosylated metabolites, 1 monohydroxylated and dehydrogenated metabolite, 2 monohydroxylated and demethylated and dehydrogenated metabolites, 2 dimerized metabolites, 3 dimerized and monohydroxylated metabolites, 2 dimerized and didemethylated metabolites, and 5 dimerized and demethylated metabolites. Finally, the major metabolic reactions of AP in rat hepatic S9 were summarized and found to be hydroxylation, demethylation, dimerization, hydration, and dehydrogenation. More importantly, the biotransformation from AP to M2 and the dimerization of AP by incubation with hepatic S9 were reported for the first time. In conclusion, this is the first report on the metabolism of a pure pterocarpan in animal tissues, and these findings will provide a solid basis for further studies on the metabolism of other pterocarpans.