Objectives Ginsenoside Rb1 is one of the major bioactive components of Panax ginseng and P. notoginseng. In the past decades, the biotransformation of Rb1 by intestinal microflora was extensively reported. However, quantitative characterization of Rb1 metabolism by human microflora is lack and effect of gender is unclear. The present study aims to quantitatively determine Rb1 metabolism by human microflora and to characterize gender difference if there is any. Methods Five independent experiments including microflora samples from 50 healthy volunteers from both genders were conducted by incubating Rb1 with microflora samples pooled from 10 individuals. Incubates were collected at 0, 2, 4, 12, 18, 24, 36, 48 and 72hr, twice extracted with butanol before HPLC analyzes. The time-course of Rb1 depletion and Rd, F2 and compound K generation were determined using an HPLC-UV method. In experiments to delineate effect of gender, Rb1 was incubated with microflora samples from 21 healthy volunteers (10 females plus 11 males) and incubates collected at 18 hr were compared. Results Metabolic patterns of Rb1 in 5 independent experiments were similar: Rb1 decreased rapidly with less than 20% of Rb1 remained intact at 12hr. Gensenoside Rd, F2 and compound K appeared in incubates in turn with Rd reaching its maximum at 12hr (represented ~50% of Rb1), followed by F2 at relatively low levels. Compound K peaked at 36hr, then kept at a constant level for another 36 hrs. A greater than 10-fold interindividual variation in Rb1 biotransformation was observed. Females exhibited the tendency to catalyze Rb1 metabolism at a higher rate. More interestingly, the metabolic pathway of Rb1 by males is the same as previously reported. However, females showed one additional pathway, in which two unknown peaks in addition to the 3 major metabolites were observed in some samples. Conclusions Gensenoside Rb1 biotransformation was quantitatively characterized for the first time. The results indicated a great interindividual variation. Females exhibited a higher metabolic capacity and an additional pathway that absent in males.