This study examined associations between MTHFR C677T polymorphism and serum folate concentrations with the risk of esophageal precancerous lesions (EPL) and esophageal squamous cell carcinoma (ESCC). The highest quartile of serum folate concentration significantly decreased the risk of ESCC compared with the lowest quartile (OR=0.11; 95% Cl, 0.04-0.33; P<0.05). MTHFR 677 C>T polymorphism was associated with the risk of ESCC by using chi-square tests (P<0.05). For the CT genotype, the risk of ESCC significantly increased in study participants with low serm folate concentrations (≤26.92 μg/L) compared with participants with high serum folate concentrations (>26.92 μg/L) by using multinomial logistic regression models. The MTHFR genotype may further modify associations between serum folate concentrations and the risk of ESCC, but it was not significantly associated with the risk of EPL.