American ginseng is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be its active constituents. We have previously demonstrated in an in vitro experiment that human enteric microbiota metabolize ginseng parent compounds into their metabolites. In this study, we analyzed American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Six healthy male volunteers ingested 1 g of American ginseng twice a day for 7 days. On day 7, biological samples were obtained and pretreated with solid phase extraction. The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 16 in feces. For the plasma, urine and feces samples, the levels of ginsenoside Rb1 (a major parent compound) were 8.6, 56.8 and 57.7 ng/mL, respectively, and the levels of compound K (a major metabolite) were 58.4 ng/mL, 109.8 ng/mL and 10.06 μg/mL, respectively. It suggested that compound K had a remarkably high level in all three samples. Moreover, in human feces, ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration. Further studies are needed to evaluate the pharmacological activities of the identified ginseng metabolites.