It has been demonstrated that vitamin C exhibits anti-cancer activity in various tumor cell lines; however, its specific mechanism of action remains unknown. Although the diagnosis and therapy of cancer patients have markedly improved in recent years, safer and more cost-effective treatments are still required. Therefore, the present study examined the effect of vitamin C on the induction of cell death in gastric cancer and its underlying mechanism of action. It was observed that the cytotoxicity of vitamin C on the human gastric cancer cell line AGS is dependent on the apoptotic pathway, including caspase cascades, but not on the necroptotic pathway. It was demonstrated that the vitamin C-induced calcium influx and ROS generation have critical roles in the induction of apoptosis. Furthermore, vitamin C treatment depleted adenosine triphosphate (ATP) production in AGS cells, and the autophagy pathway may be involved in this process. Taken together, the current study suggests that a high dose of vitamin C may induce gastric cancer cell apoptosis through the dysfunction of mitochondria, including calcium influx, reactive oxygen species generation and ATP depletion.