Objective To explore the association between methylenetetrahydrofolate reductase (MTHFR) rs1801133 polymorphism and lung cancer risk. Methods This case-control study included 974 lung cancer patients and 1 005 healthy controls living in Shanghai and Taizhou, Jiangsu Province. After collecting the peripheral blood samples of the participants, the whole blood genomic DNA was extracted for MTHFR gene rs1801133 genotyping. The effect of rs1801133 on lung cancer susceptibility was analyzed through unconditional logistic regression analysis. Results Compared with CC genotype, MTHFR rs1801133 CT and TT genotypes significantly decreased lung cancer risk (odds ratio[OR]=0.801, 95% confidence interval[CI]:0.651-0.985, P=0.035; OR=0.754, 95% CI:0.582-0.975, P=0.032), but this association became insignificant after adjusting age, gender, smoking status, and family cancer history (OR=0.841, 95% CI:0.677-1.045, P=0.118; OR=0.799, 95% CI:0.609-1.047, P=0.104). However, in dominant model, CT+TT genotypes exhibited a significantly reduced lung cancer risk in males (OR=0.764, 95% CI:0.597-0.977, P=0.032) and those with a family cancer history (OR=0.600, 95% CI:0.385-0.925, P=0.022), and a significantly reduced risk for having squamous cell carcinoma (OR=0.727, 95% CI:0.542-0.976, P=0.033). Conclusion The MTHFR gene rs1801133 C>T mutation might reduce the risk of lung cancer in males and those with a family history of cancer, especially those with squamous cell carcinoma.