Abstract: In order to understand the effect of dietary components on the absorption of ginsenosides and their metabolites into the blood, we studied the pharmacokinetics of the ginseng extract and its main constituent ginsenoside Rb1 in rats with or without pretreatment with a prebiotic fiber, NUTRIOSE®, by liquid chromatography tandem mass spectrometry (LC–MS/MS). When ginsenoside Rb1 was incubated with rat feces, its main metabolite was ginsenoside Rd. When the intestinal microbiota of rat feces were cultured in vitro, their ginsenoside Rd-forming activities were significantly induced by NUTRIOSE®. When ginsenoside Rb1 was orally administered to rats, the maximum plasma concentration (Cmax) and area under the plasma drug concentration time curve (AUC) for the main metabolite, ginsenoside Rd, was 72.4 ± 31.6 ng/mL and 663.9 ± 285.3 μg·h/mL, respectively. When the ginseng extract (2,000 mg/kg) was orally administered, Cmax and AUC for ginsenoside Rd were 906.5 ± 330.2 ng/mL and 11,377.3 ± 4,470.2 μg·h/mL, respectively. When ginseng extract was orally administered to rats fed NUTRIOSE® containing diets (2.5%, 5%, or 10%), Cmax and AUC were increased in the NUTRIOSE® receiving groups in a dose-dependent manner. These findings reveal that intestinal microflora promote metabolic conversion of ginsenoside Rb1 and ginseng extract to ginsenoside Rd and promote its absorption into the blood in rats. Its conversion may be induced by prebiotic diets such as NUTRIOSE®.