Multidrug resistance has became the major obstacle to cancer chemotherapy. Recent studies suggest that quercetin could enhance the response of tumors to chemotherapy although the mechanism by which quercetin enhances the sensitivity of tumor cells to chemical drugs remains elusive. Therefore, in this study, we examined the effects of quercetin on doxorubicin cytotoxicity in human breast cancer cells and investigated the underlying mechanisms. MCF-7 and MCF-7/ dox cells were exposed to doxorubicin, quercetin, or combination of both agents for 36 hours. Cell proliferation, cell invasion, intracellular doxorubicin concentration and expression of hypoxia-inducible factor-1 alpha (HIF-1α) and P-glycoprotein (P-gp) were then assessed. Quercetin had little effect on cell proliferation at concentrations less than 0.7 μM. Compared to treatment with doxorubicin alone, combined treatment with doxorubicin and quercetin (0.7 μM) significantly inhibited cell proliferation and invasion and suppressed the expression of HIF-1α and P-gp. Quercetin (0.7 μM) increased the intracellular doxorubicin concentration and enhanced doxorubicin cytotoxicity as 1.49-fold in MCF-7 cells and 1.98-fold in MCF-7/dox cells. These data suggest that quercetin can increase the chemosensitivity of breast cancer cells to doxorubicin.