P-glycoprotein (P-gp) is an ATP-dependent efflux transporter highly expressed in gastrointestinal tract and multidrug resistance tumor cells. Inhibition or induction of P-gp can cause drug-drug interactions and thus influence the effects of P-gp substrate drugs. Previous studies indicated that 20(S)-ginsenoside Rh2 [20(S)-Rh2] could synergistically enhance the anticancer effects of conventional chemotherapeutic agents at a nontoxic dose. The aim of present study was to investigate in vitro and in vivo whether 20(S)-Rh2 was a P-gp inhibitor and analyze the possible inhibitory mechanisms and potential herb-drug interactions. Results showed that in vitro, 20(S)-Rh2 significantly enhanced rhodamine 123 retention in cells and decreased the efflux ratio of digoxin, fexofenadine, and etoposide, which were comparable to the effects of the established P-gp inhibitor verapamil. However, the transport of 20(S)-Rh2 suggested that 20(S)-Rh2 was not a P-gp substrate. Furthermore, the inhibitory effect persisted for at least 3 h after removal of 20(S)-Rh2. Unlike P-gp substrates, 20(S)-Rh2 inhibited both basal and verapamil-stimulated P-gp ATPase activities. It also significantly decreased UIC2 binding fluorescence, a marker for conformational change of P-gp. In situ and in vivo experiments showed that 20(S)-Rh2 increased the area under the plasma concentration-time curve and maximum plasma concentration of digoxin, fexofenadine, and etoposide significantly without affecting terminal elimination half-time. Long-term treatment with 20(S)-Rh2 failed to affect intestinal P-gp expression in vitro and in vivo. In conclusion, 20(S)-Rh2 is a potent noncompetitive P-gp inhibitor, which indicates a potential herb-drug interaction when 20(S)-Rh2 is coadministered with P-gp substrate drugs. It could increase the absorption of P-gp substrate drugs without long-term induction of P-gp expression in rats.