Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in some, but not all cancer cells. To assess the regulation of TRAIL-resistance in the human gastric cancer cells, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. All the gastric cancer cell lines tested were susceptible to TRAIL to some extent, except for SNU-216 cell line, which was completely resistant. TRAIL receptor expression was not related to the TRAIL-sensitivity. Of the cell lines tested, SNU-216 showed the highest level of constitutively active Akt and the short form of FLICE inhibitory protein (FLIP(S)). Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or with the protein synthesis inhibitor cycloheximide induced a suppression of constitutive Akt activation in SNU-216 cells and a concomitant decrease in the expression of FLIP(S). The reduction of Akt activity by LY294002 affected the transcriptional level of FLIP(S), but not the mRNA stability. As a result, LY294002 or cycloheximide significantly enhanced TRAIL-induced apoptosis. Moreover, the overexpression of constitutively active Akt in the TRAIL-sensitive cell line, SNU-668, rendered the cell line resistant to TRAIL. In addition, infection of the same cell line with retrovirus expressing FLIP(S) completely inhibited TRAIL-induced apoptosis by blocking the activation of caspase-8. Therefore, our results suggest that Akt activity promotes human gastric cancer cell survival against TRAIL-induced apoptosis via upregulation of FLIP(S), and that the cytotoxic effect of TRAIL can be enhanced by modulating the Akt/FLIP(S) pathway in human gastric cancers.