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Abstract: Objective To explore the reversal effect of (-)-5-N-acetylardeemin on adriamycin resistance in multidrug-resistant cancer cells including human breast cancer cells MCF-7/Adr and human non-small cell lung cancer cells A549/Adr in vitro. Methods The multidrug-resistant cancer cells MCF-7/Adr, A549/Adr and their respective parental cells were treated with different concentrations of (-)-5-N-acetylardeemin and adriamycin individually or in combination. Cell death was detected based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. Intracellular accumulation of adriamycin was measured by the detection of fluorescence intensity of cell lysates using microplate reader. The expression of P-glycoprotein (P-gp) was evaluated by Western blot. Results (-)-5-N-acetylardeemin significantly reversed the adriamycin resistance in MCF-7/Adr and A549/Adr in a dose-dependent manner, and the reversal folds were 10.8 in MCF-7/Adr cells and 20.1 in A549/Adr cells with the treatment of 10 μmol/L (-)-5-N-acetylardeemin. (-)-5-N-acetylardeemin also enhanced the sensitivity of parental MCF-7 and A549 cells to adriamycin. The fluorescence intensity in both MCF-7/Adr and A549/Adr cells, which reflected the intracellular accumulation of adriamycin, were significantly enhanced by (-)-5-N-acetylardeemin in a dose-dependent manner. The expressions of P-gp in MCF-7/Adr and A549/Adr cells were significantly inhibited by (-)-5-N-acetylardeemin. Conclusion (-)-5-N-acetylardeemin could reverse the multidrug resistance in cancer cells through inhibiting the expression of P-gp and enhancing the intracellular accumulation of cytotoxic drug.