Human gastric cancer cells are generally believed to be less sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, but the events responsible for this resistance are as yet unclear. In this study, we investigated the role of the phosphoinositide 3-kinase (PI3K)/Akt, nuclear factor κB (NF-κB) and extracellular signal-regulated kinase (ERK) signaling pathways in the TRAIL resistance of gastric cancer cells. TRAIL failed to induce observable apoptosis in the three cell lines. Further investigation revealed that TRAIL engagement led to the activation of PI3K/Akt as well as of NF-κB. The inhibition of PI3K/Akt by a specific inhibitor facilitated TRAIL-induced apoptosis. Blockage of TRAIL-induced NF-κB activation by transient transfection with a phosphorylation-defective mutant IκB also enhanced the sensitivity of cells towards TRAIL. Meanwhile, ERKs were highly activated in the resting cells and were not further activated by TRAIL treatment. However, the inhibition of ERK activity by PD98059 also enhanced the apoptosis-inducing ability of TRAIL. Our data demonstrated that the activation of PI3K/Akt and NF-κB by TRAIL is responsible for resistance to TRAIL in human gastric cancer cells. Blockage of survival signals significantly enhances the apoptosis induced by TRAIL.