We examined the relationship between the C825T, C1429T, and A-350G variants in the G-protein beta 3 subunit (GNB3) gene, the A1330V and Q89R variants in the low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the risk of hypercholesterolemia in a prospective study in Japanese workers. This study included observations over a 5-year period from 1997 to 2002 on 936 males and 662 females who were not hypercholesterolemic on entry. Hypercholesterolemia was defined as a serum total cholesterol level of 240 mg/dL or higher. Pooled logistic regression analyses were performed using either of the gene variants with age, body mass index, smoking, alcohol consumption, and habitual exercise as the covariates. The risk of the development of hypercholesterolemia was 2.27 times higher in males with the TT genotype of GNB3/C1429T than in males with the CC genotype (95% confidence interval, 1.04-4.94), after adjustment for the effects of other potential covariates. Simultaneously, the risk was 1.49 times higher in males with the AV genotype of LRP5/A1330V than in males with the AA genotype (95% confidence interval, 1.04-2.12) after adjustment for the effects of other potential covariates. This study indicates the GNB3/C1429T and LRP5/A1330V are independent risk factors for hypercholesterolemia in Japanese males and suggests that targeting these polymorphisms may be beneficial when attempting to prevent hypercholesterolemia in the general Japanese male population.