Several genome-wide association studies (GWAS) have revealed that single nucleotide polymorphisms (SNPs) of ABCG2 and SLC22A12 were strongly associated with serum uric acid (SUA), but those of methylene tetrahydrofolate reductase (MTHFR) were not. However, there were several studies indicating the association with MTHFR C677T polymorphism. This study examined the association with the polymorphism, taking into account the genotypes of ABCG2 Q126X and SLC22A12 W258X. Subjects were 5,028 health checkup examinees of Seirei Preventive Health Care Center (3,416 males and 1,612 females) aged 35 to 69 years, who participated in the Japan Multi-Institutional Collaborative Cohort Study (J-MICC Study). Hyperuricemia was defined as SUA equal to 7 mg/dL or over. The genotype frequency was 35.9% for CC, 48.1% for CT, and 16.0% for TT, being in Hardy-Weinberg equilibrium (p=0.90). Among 4,425 participants with ABCG2 126QQ and SLC22A12 258WW who were not under medication for hyperuricemia, the mean SUA was 5.6 mg/dL, 5.6 mg/dL, and 5.7 mg/dL, respectively. When 114 participants with ABCG2 126QQ and SLC22A12 258WW under medication for hyperuricemia were included in hyperuricemia cases, the sex-age adjusted odds ratio (OR) of hyperuricemia was not significant; OR=1.00 (95% confidence interval, 0.89-1.24) for CT genotype and OR=0.98 (0.84-1.32) for TT genotype, relative to CC genotype. The present study indicated no association between SUA and MTHFR C677T genotype, after the influences of ABCG2 Q126X and SLC22A12 W258X were removed.