Cellular prion protein (PrP(C)), a glycosyl-phosphatidylinositol-anchored membrane protein with unclear physiological function, was previous found to be upregulated in adriamycin (ADR)-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Overexpression of PrP(C) in gastric cancer has certain effects on drug accumulation through upregulation of P-glycoprotein (P-gp), which is suggested to play an important role in determining the sensitivity of tumor cells to chemotherapy and is linked to activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. In the present study, we further investigate the role of the PI3K/Akt pathway in PrP(C)-induced multidrug-resistance (MDR) in gastric cancer. Immunohistochemistry and confocal microscope detection suggest a positive correlation between PrP(C) and phosphorylated Akt (p-Akt) expression in gastric cancer. Using established stable PrP(C) transfectant cell lines, we demonstrated that the level of p-Akt was increased in PrP(C)-transfected cells. Inhibition of PrP(C) expression by RNA interference resulted in decreased p-Akt expression. Inhibition of the PI3K/Akt pathway by one of its specific inhibitors, LY294002, or by Akt small interfering RNA (siRNA) resulted in decreased multidrug resistance of SGC7901 cells, partly through downregulation of P-gp induced by PrP(C). Taken together, our results suggest that PrP(C)-induced MDR in gastric cancer is associated with activation of the PI3K/Akt pathway. Inhibition of PI3K/Akt by LY2940002 or Akt siRNA leads to inhibition of PrP(C)-induced drug resistance and P-gp upregulation in gastric cancer cells, indicating a possible novel mechanism by which PrP(C) regulates gastric cancer cell survival.