Objective: To investigate the reversal effect of the PI3K/Akt singling pathway inhibitor MK-2206 on drug resistance in human breast cancer cells and its mechanism. Methods: The inhibitory effects of doxorubicin and MK-2206 on growth of MCF-7 cells (doxorubicin-sensitive) and MCF-7/ADR cells (doxorubicin-resistant) were examined by CCK-8 assay and their IC50 values were calculated. Based on the IC50 results, MCF-7 and MCF-7/ADR cells were treated with a selected low-toxic concentration of doxorubicin alone or combined with different concentrations of MK-2206, and then the influence of MK-2206 on doxorubicin-resistance and doxorubicin-induced apoptosis, and on doxorubicin accumulation in MCF-7/ADR cells were detected by CCK-8 assay and flow cytometry, respectively. MCF-7 and MCF-7/ADR cells were treated with MK-2206 alone, and then the expressions of the proteins associated with PI3K/Akt signaling pathway were determined by Western blot. Results: The proliferations in both types of cells were significantly inhibited by either doxorubicin or MK-2206 treatment, and the IC50 value of doxorubicin for MCF-7/ADR cells was significantly higher than that for MCF-7 cells (P<0.05), while the IC50 values between MK-2206 for the both type of cells had no significant difference (P>0.05). The IC50 values of doxorubicin for both types of cells were decreased by combination treatment of MK-2206, but the decreasing degree in MCF-7/ADR cells was significantly greater than that in MCF-7 cells (P<0.05). MK-2206 exerted no obvious influence on the doxorubicin-induced apoptosis in MCF-7 cells, but significantly increased the doxorubicin-induced apoptosis in MCF-7/ADR cells (P<0.05). No significant difference was noted in doxorubicin accumulation among MCF-7/ADR cells treated with various concentrations of MK-2206 (P>0.05). After MK-2206 treatment alone, the protein expression of p-(Thr308) Akt was significantly down-regulated (P<0.05), and the protein expression of p-(Thr246) PRAS40 showed no significant change (P>0.05) in MCF-7 cells, while both of the above protein expressions were significantly down-regulated in MCF-7/ADR cells (both P<0.05). Conclusion: MK-2206 can partially reverse the drug resistance in breast cancer cells via inhibition of the PI3K/Akt signaling pathway, in which PRAS40 protein activation may contribute importantly to the drug resistance in breast cancer cells.