Ginsenoside Rb1 is an active protopanaxadiol saponin from Panax species. In order to compare the similarities and differences of microbial and mammalian metabolisms of ginsenoside Rb1, the microbial transformation by Acremonium strictum and metabolism in rats were comparatively studied. Microbial transformation of ginsenoside Rb1 by Acremonium strictum AS 3.2058 resulted in the formation of eight metabolites. Ten metabolites (M1-M10) were detected from the in vivo study in rats and eight of them were identified as the same compounds as those obtained from microbial metabolism by liquid chromatography-tandem mass spectrometry analysis and comparison with reference standards obtained from microbial metabolism. Their structures were identified as ginsenoside Rd, gypenoside XVII, 20(S)-ginsenoside Rg3, 20(R)-ginsenoside Rg3, ginsenoside F2, compound K, 12beta-hydroxydammar-3-one-20(S)-O-beta-d-glucopyranoside, and 25-hydroxyl-(E)-20(22)-ene-ginsenoside Rg3, respectively. The structures of the additional two metabolites were tentatively characterized as 20(22),24-diene-ginsenoside Rg3 and 25-hydroxyginsenoside Rd by HPLC-MS/MS analysis. M7-M10 are the first four reported metabolites in vivo. The time course of rat metabolism of ginsenoside Rb1 was also investigated.