Multidrug resistance(MDR) is a major obstacle to efficiency of breast cancer chemotherapy. We investigated whether combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells and tried to elucidate the possible mechanisms. The combination of metformin and 2-deoxyglucose selectively enhanced cytotoxicity of doxorubicin against MCF-7/Dox cells. Combination of the two drugs resumed p53 function via inhibiting overexpression of murine doubleminute 2(MDM2) and murine doubleminute 4(MDM4) leading to G2/M arrest and apoptosis in MCF-7/Dox cells. Combination of the two drugs had no effect on P-glycoprotein mRNA expression and P-glycoprotein ATPase activity but increased doxorubicin accumulation in MCF-7/Dox cells. The increased doxorubicin accumulation maybe associate with metabolic stress. Combination of metformin and 2-deoxyglucose initiated a strong metabolic stress in MCF-7/Dox cells via inhibiting glucose uptake, lactate, fatty acid, ATP production and protein kinase B(AKT)/ mammalian target of rapamycin(mTOR) pathway. Taken together, combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells by recovering p53 function and increasing doxorubicin accumulation. Furthermore, doxorubicin selectively increases MCF-7/Dox apoptosis via aggravating metabolic stress induced by metformin plus 2-deoxyglucose. The mutually reinforcing effect made the combination of metformin and 2DG had a better effect on reversing MDR.