Background: Bacterial conversion of ginsenosides is crucial for the health-promoting effects of ginsenosides. Previous studies on the biotransformation of ginsenoside Rb1 (Rb1) by gut bacteria have focused on the ginsenoside Rd (Rd) pathway (Rb1 → Rd → ginsenoside F2 (F2) → compound K (Cpd K)). This study aims to examine the gypenoside pathway in human gut bacteria in vitro.Methods: The metabolic pathways of ginsenoside Rb1 and its metabolites ginsenoside Rd and gypenoside XVII in human gut bacteria were investigated by incubating the compounds anaerobically with pooled or individual gut bacteria samples from healthy volunteers. Ginsenoside Rb1, the metabolites generated by human gut bacteria, and degraded products in simulated gastric fluid (SGF) were qualitatively analyzed using an LC/MSD Trap system in the negative ion mode and quantitatively determined by HPLC-UV analysis.Results: When incubated anaerobically with pooled gut bacteria, Rb1 generated five metabolites, namely Rd, F2, Cpd K, and the rare gypenosides XVII (G-XVII) and LXXV (G-LXXV). The gypenoside pathway (Rb1 → G-XVII → G-LXXV → Cpd K) was rapid, intermediate, and minor, and finally converted Rb1 to Cpd K via G-XVII → F2 (major)/G-LXXV (minor). Both the Rd and gypenoside pathways exhibited great inter-individual variations in age-and sex-independent manners (P > 0.05). Rb1 was highly acid-labile and degraded rapidly to form F2, ginsenoside Rg3, ginsenoside Rh2, and Cpd K, but did not generate the gypenosides in SGF. The formation of the gypenosides might be explained by the involvement of a gut bacteria-mediated enzymatic process.Conclusions: Rb1 was metabolized to G-XVII, F2 (major) or G-LXXL (minor), and finally Cpd K by human gut bacteria in vitro. © 2013 Shen et al.; licensee BioMed Central Ltd.