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Background It has been demonstrated that platycodin D (PD) exhibits anti-cancer activities. This study aims to investigate the anti-proliferative effects of the combination of PD and doxorubicin (DOX) on human breast cancer cells (MCF-7 and MDA-MB-231 cells). Methods The anti-proliferative effects of different dosages of PD, DOX, and PD + DOX on MCF-7 and MDA-MB-231 cells were determined by the MTT assay. The 10 μM PD, 5 μM DOX, and 10 μM PD + 5 μM DOX induced-protein expression of apoptosis-related molecules on MCF-7 and MDA-MB-231 cells were detected by western blot. The 10 μM PD, 5 μM DOX and 10 μM PD + 5 μM DOX-induced mitochondrial membrane potential changes on MCF-7 and MDA-MB-231 cells were stained with JC-1 before visual determination. The intracellular accumulations of DOX, induced by 10 μM PD, 5 μM DOX and 10 μM PD + 5 μM DOX, were detected by flow cytometry. Results PD enhanced anti-cancer activities of DOX were observed in both MCF-7 and MDA-MB- 231 cell lines. Compared with mono treatment, the combined treatment increased the protein expression of cleaved poly (ADP-ribose) polymerase and decreased the mitochondrial membrane potential. The combined treatment with PD did not obviously increase the accumulation of DOX in MCF-7 cells (1.66 ± 0.13 in DOX-treated group, and 1.69 ± 0.06 in PD + DOX-treated group, P = 0.76), but it significantly increased the accumulation of DOX in MDA-MB-231 cells (1.76 ± 0.17 in DOX-treated group, 2.09 ± 0.02 in PD + DOX-treated group, P = 0.027). Conclusion The combined treatment of DOX and PD exhibited stronger anti-proliferative effects on MCF-7 and MDA-MB-231 cells than DOX and PD treatment did. [ABSTRACT FROM AUTHOR]