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The results of prospective studies that have examined the association between serum cholesterol levels and the incidence of gastric cancer remain controversial. To examine this issue in a general population, a total of 2,604 subjects aged 40 years or older were followed up prospectively for 14 years. During the follow-up period, gastric cancer developed in 97 subjects. The age- and sex-adjusted incidence of gastric cancer by quartiles of serum cholesterol level, namely, <4.06, 4.06–5.32, 5.33–6.04 and ≥6.05 mmol/L, were 3.9, 3.3, 3.1 and 2.1 per 1,000 person–years, respectively. The risk of gastric cancer increased with decreasing cholesterol level (age- and sex-adjusted hazard ratio [HR], 1.22; 95% confidence interval [CI], 1.01–1.49;p= 0.04 for a decrease of 1 mmol/L in serum cholesterol level). This inverse association remained unchanged even after adjustment for other confounding factors, namely,Helicobacter pyloriinfection, atrophic gastritis, family history of malignant neoplasm, smoking habits, body mass index, hemoglobin A1c, white blood cell count and dietary factors (adjusted HR, 1.28; 95% CI, 1.03–1.58;p= 0.02). This association was significant for intestinal-type gastric cancers, but not for diffuse-type. As regards cancer stage, the inverse cholesterol-cancer association was marginally significant for early gastric cancer after multivariate-adjustment (adjusted HR, 1.25; 95% CI, 0.97–1.61;p= 0.09), but was not for advanced gastric cancer probably due to the small number of cases. In conclusion, our findings suggest that low serum cholesterol levels are an independent risk factor for developing gastric cancer, especially intestinal-type gastric cancer.