The major obstacle to successful treatment of gastric cancer is chemotherapy resistance. Our study was designed to investigate the role of phosphoinositide 3‐kinase (PI3K)/Akt pathway in the development of chemoresistance in gastric cancer. In the present study, elevated Akt expression and Akt phosphorylation (Ser 473), as well as decreased PTEN expression were observed in 28 cases of gastric cancer tissues. Etoposide and doxorubicin stimulated Akt and PI3K activities in 2 gastric cancer cell lines (BGC‐823 and SGC‐7901), and the activities were concentration and time‐dependent. Up‐regulation of PTEN expression in BGC‐823 cells by PEAK8‐PTEN transient transfection obviously decreased the basal and anticancer drugs induced Akt activities, then sensitized BGC‐823 cells to etoposide and doxorubicin. Pretreatment of BGC‐823 and SGC‐7901 cells with wortmannin, a PI3K inhibitor, attenuated cells's resistance to etoposide and doxorubicin. In addition, pretreatment of wortmannin blocked etoposide and doxorubicin induced IkappaB‐α degradation, NFkappaB activation, phosphorylation of Akt, MDM‐2 and forkhead transcription factors. Wortmannin pretreatment also promoted the accumulation of p27/Kip, but inhibited the Mcl‐1 expression. Furthermore, wortmannin promoted etoposide and doxorubicin induced caspase‐3, caspase‐9 activation and poly ADP‐ribose polymerase cleavage. Taken together, the observations indicate the PI3K/Akt pathway plays an important role in the chemoresistance of gastric cancer cells. A new strategy for combined chemotherapy of gastric cancer should be designed to more specifically block PI3K/Akt pathway and then decrease the amount of resistant cells. © 2007 Wiley‐Liss, Inc.