One of the major causes for cancer cells to resist current chemotherapy is attributed to the over-expression of P-glycoprotein (P‐gp), resulting in insufficient drug delivery to the tumor sites. Protopanaxadiol ginsenosides Rg3 and Rh2 are known to induce apoptosis and significantly enhance the tumor inhibitory effects of chemotherapeutics in a synergistic fashion. One of the possible mechanisms is by blocking P‐gp activity. The final deglycosylation metabolite of protopanaxadiols (PPDs) in vivo is 20(S)-protopapanaxadiol (aglycone PPD, aPPD), which has also shown anticancer activity and synergy with chemotherapy drugs. In the present study, P‐gp over-expressing cancer cells were utilized to test whether aPPD also inhibits P‐gp activity. We found that aPPD caused similar cytotoxicity in P388adr cells as their parental non-MDR cells, suggesting that aPPD may not be a substrate of P‐gp. On the other hand, the calcein AM efflux assay showed that aPPD was able to inhibit P‐gp activity as potently as verapamil on MDR cells. The blockage of P‐gp activity was highly reversible as wash-out of aPPD resulted in an immediate recovery of P‐gp activity. Unlike verapamil, aPPD did not affect ATPase activity of P‐gp suggesting a different mechanism of action. The above results indicate that aPPD, unlike its precursor ginsenosides Rg3 and Rh2, is not a substrate of P‐gp. It is also the first time that aPPD has showed a reversible nature of its P‐gp inhibition. In addition to its pro-apoptotic nature, aPPD may be a potential new P‐gp inhibitor for cancer treatment.