Methylenetetrahydrofolate reductase (MTHFR) catalyzes the metabolism of folate and nucleotides needed for DNA synthesis and repair. Variations in MTHFR functions likely play roles in the etiology of lung cancer. The MTHFR gene has three nonsynonymous single nucleotide polymorphisms (i.e., C677T, A1298C, and G1793A) that have a minor allele frequency of >5%. We investigated the associations between the frequencies of MTHFR variant genotypes and risk of lung cancer in a hospital-based case-control study of 1,051 lung cancer patients and 1,141 cancer-free controls in a non-Hispanic White population. We found that compared with the MTHFR 1298AA genotype, the 1298CC genotype was associated with a significantly increased risk of lung cancer in women [(odds ratio (OR), 2.09; 95% confidence interval (95% CI), 1.32-3.29)] but not in men (OR, 0.95; 95% CI, 0.62-1.45). The MTHFR 677TT genotype was associated with a significantly decreased risk of lung cancer in women (OR, 0.60; 95% CI, 0.40-0.92) but not in men. No association was found between the MTHFR G1793A polymorphism and risk of lung cancer. Further analysis suggested evidence of gene-dietary interactions between the MTHFR C677T polymorphism and dietary intake of vitamin B6, vitamin B12, and methionine in women and evidence of gene-environment interactions between the MTHFR C677T and A1298C polymorphisms and tobacco smoking in men. In conclusion, the polymorphisms of MTHFR may contribute to the risk of lung cancer in non-Hispanic Whites and modify the risk associated with the dietary and environmental exposure in a sex-specific manner.