Phosphatidylinositol 3-kinase/AKT1 pathway was an important intracellular pathway that was frequently activated in cancer cells. In the present study, we constructed the siRNA eukaryotic expression vectors of AKT1 and transfected them into AGS cells to examine whether the down-regulation of AKT1 increased cell sensitivity towards chemotherapeutic drugs. After transfection, the expression of AKT1 was dramatically decreased in AKT1 siRNA transfectants compared with that in parental cells and empty vector control cells. The down-regulation of AKT1 could significantly enhance the sensitivity of AGS cells to vincristine, adriamycin, 5-fludrouracil and cisplatin. AKT1 siRNA could significantly down-regulate the expression of Bcl-2, and up-regulate the expression of Bax, but not alter the expression of PTEN in gastric cancer cells. These observations suggested that the siRNA constructs of AKT1 we obtained could effectively down-regulate the expression of AKT1 and reverse the resistant phenotype of gastric cancer cells. The further study of the biological functions of AKT1 may be helpful for understanding the mechanisms of multidrug resistance of gastric cancer and developing possible strategies to treat gastric cancer.