The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been used to treat a variety of cancer cells. However, since some gastric cancer cells are resistant to TRAIL, we explored whether reovirus induces cytolysis in TRAIL-resistant gastric cancer cells. We found that TRAIL-resistant SNU-216 gastric cancer cells were susceptible to apoptosis by reovirus infection. Furthermore, co-treatment with reovirus and TRAIL accelerated apoptosis of SNU-216 cells by down-regulation of Akt activation as assessed by a very low activation of Akt in TRAIL-sensitive SNU-668 gastric cancer cells. Inhibition of Akt signaling with wortmannin or suppression of Akt expression with sh-Akt lentivirus promoted reovirus-mediated apoptosis of SNU-216 gastric cancer cells. Reovirus infection also down-regulates the activation of signaling molecules such as Ras and ERK involved in cell proliferation and survival but not the activation of p38 MAPK involved in cellular stress. In addition, the co-treatment with reovirus and TRAIL resulted in cleavage of caspase-8, caspase-9 and Bid, leading to a decrease in the mitochondrial membrane potential, indicating that reovirus may utilize the mitochondrial intrinsic apoptotic pathway in TRAIL-resistant SNU-216 gastric cancer cells. Accordingly, we first demonstrate that reovirus infection down-regulates Akt activation, leading to apoptosis of TRAIL-resistant gastric cancer cells.