Gastric cancer is one of the most common malignant tumors and the second cause of cancer-related deaths worldwide. Naphthoquinones such as juglone and plumbagin are compounds used extensively to overcome resistance to chemotherapeutic agents in cancers due to their cytotoxic role. This study is the first to investigate the anti-cancer effect of naphthazarin (Naph), one of the naphthaquinones, in human gastric cancer AGS cells. We showed that Naph exhibited effective preferential cell growth inhibition via G2/M phase arrest and apoptosis, which was associated with reduced levels of Cdc2 and Cdc25C expression. Naph also increased cleaved caspase-3 and Poly ADR(adenosine diphosphate ribose) Polymerase expression, γ-H2AX expression (an indicator of DNA double strand breaks) and DNA fragmentation. We also found the generation of reactive oxygen species is a critical mediator in Naph-induced cell growth inhibition and apoptosis. The non-protein antioxidant, glutathione significantly abolished Naph-mediated inhibition of cell growth and apoptosis. Taken together, our findings showed that Naph not only inhibited cell growth, but also induced apoptosis of AGS cells, suggesting that Naph may be a potential candidate for cancer therapy against gastric cancers.