Oxidative stress plays a critical role in apoptosis of gastric epithelial cells. Omega-3 fatty acids show anti-inflammatory and/or anticancer effects and regulate apoptosis in various cells. In this study, we aimed to investigate whether omega-3 fatty acids inhibit oxidative stress-induced apoptosis of gastric epithelial cells. The cells received oxidative stress caused by silica-immobilized glucose oxidase acting on β-d-glucose and cultured in the absence or presence of α-linolenic acid or docosahexanoic acid. Viable cell numbers, levels of H2O2 in the medium, DNA fragmentation, and protein levels of p53 and Bax were determined. As a result, silica-immobilized glucose oxidase acting on β-d-glucose consistently and reproducibly produced H2O2, which decreased cell viability and increased DNA fragmentation of the cells. Omega-3 fatty acids inhibited oxidative stress-induced cell death, DNA fragmentation, and induction of p53 and Bax of the cells. The silica-immobilized glucose oxidase could be a useful tool for studies on oxidative stress-induced cellular events because it is reusable and forms a stable enzyme system acting on glucose. Omega-3 fatty acids may be beneficial for preventing oxidative stress-induced apoptosis by inhibiting apoptotic gene expression and DNA fragmentation of gastric epithelial cells.