Diallyl trisulfide (DATS), one of the principal constituents of garlic oil, is a kind of organosulfur compound with high anti-cancer activity. Although inhibition of cancer cell proliferation by DATS is known to be associated with the induction of apoptosis and cell cycle arrest related to reactive oxygen species (ROS) production, it is still necessary to study the detailed mechanisms. In this study, we investigated the role of ROS on the activation of AMP-activated protein kinase (AMPK) in DATS-induced apoptosis and cell cycle arrest in AGS human gastric carcinoma cells. The results of the present study indicate that DATS inhibited proliferation of AGS cells by promoting apoptosis, and accumulating cellular portion of G2/M phase via the induction of cyclin B1 and cyclin-dependent kinase p21(WAF1/CIP1). The phosphorylation of histone H3 was also markedly increased following treatment with DATS, revealing that DATS stimulated a mitotic arrest, not the G2 phase. Furthermore, we found that DATS concurrently induced phosphorylation of AMPK; however, chemical inhibition of AMPK by compound C, an AMPK inhibitor, significantly blocked apoptosis induced by DATS, suggesting that DATS induces cytotoxicity of AGS cells through the AMPK-dependent pathway. Moreover, DATS provoked intracellular ROS generation and the loss of mitochondrial membrane potential, and in particular, when ROS production was blocked by antioxidant N-acety-l-cysteine, both AMPK activation and growth inhibition by DATS were completely abolished. Collectively, these findings suggest that DATS inhibited growth of AGS cells, which was mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis, and cell cycle arrest, through a ROS-dependent activation of AMPK pathway.