Despite conflicting results, there is evidence to suggest an inverse link between total body cholesterol levels and the risk of certain malignancies. Based on previous reports, this phenomenon appears to vary with cancer site, and, in particular, more consistent data on inverse relations was reported in the risk of gastric cancer. In the current study, the effect of cholesterol on gastric cancer cell viability was examined using an in vitro cell culture system. Addition of cholesterol in culture medium resulted in reduced viability and clonogenicity of SNU601, SNU638 and SNU216 gastric cancer cells by induction of both autophagic and apoptotic death. Transient inactivation of ERK1/2 was linked to reduction of cholesterol-mediated cell viability, and tumor necrosis factor‑related apoptosis-inducing ligand receptor 2 (TRAIL‑R2/DR5) was also involved in cell death signaling. In conclusion, these results imply that cholesterol can act as a signal regulator to modulate cell viability and that proper cellular cholesterol levels may be advantageous to suppress growth of gastric carcinomas.