〔Abstract〕 Objective To explore the mechanism of Akt inhibitor MK-2206 in enhancing the sensitivity of pancreatic cancer cells to gemcitabine under hypoxia. Methods Hypoxic microenvironment was established using physical hypoxiain vitro. The pancreatic cancer cell lines were cultured under hypoxia conditions(oxygen concentration 1%) for 48 h, and cancer cells cultured under normoxia(oxygen concentration 21%) were used as control. Western blot was used to detect the expression changes of hypoxia inducible factor-1α(HIF-1α), p-Akt(Ser473) and Akt in pancreatic cancer cells under normoxia and hypoxia. Effect of MK-2206 on p-Akt(Ser473) of pancreatic cancer cells(MIA PaCa-2 and BxPC-3) was detected by Western blot under nomoxia and hypoxia. Effect of MK-2206 on gemcitabine sensitivity of pancreatic cancer cells(MIA PaCa-2 and BxPC-3) was investigated by CCK-8 cytotoxicity test under hypoxia. Results The results of Western blot showed that hypoxia microenvironment promoted accumulation of HIF-1α, as well as increased phosphorylation of Akt(Ser473) in pancreatic cancer cells. In addition, MK-2206 inhibited p-Akt(Ser473) in pancreatic cancer cells under normoxia and hypoxia. Cytotoxicity test results showed that the relative cell viability of co-treatment with MK-2206(1 μmol/L) and gemcitabine(10 μmol/L) group(0.549±0.001) was lower than that of co-treatment with DMSO and gemcitabine(10 μmol/L) group(0.632±0.017) in MIA PaCa-2 cells under hypoxia, and the difference was statistically significant(t=12.107,P=0.007). Furthermore, the relative cell viability of co-treatment with MK-2206(1 μmol/L) and gemcitabine(10 μmol/L) group(0.508±0.041) was lower than co-treatment with DMSO and gemcitabine(10 μmol/L) group(0.594±0.106) in BxPC-3 cells under hypoxia, and the difference was statistically significant(t=5.363,P=0.033). Conclusion MK-2206 could enhance the sensitivity of pancreatic cancer cells to gemcitabine under hypoxic microenvironment.