Methylenetetrahydrofolate reductase (MTHFR) is one of the important enzymes involved in folate metabolism. A single (C→T) substitution at nucleotide 677 of MTHFR gene influences the enzyme activity and is correlated with susceptibility to several tumour types. In order to compare the association of the MTHFR C677T polymorphism with susceptibility to esophageal squamous cell carcinoma (ESCC) in a Northern Chinese population, high-speed real-time PCR and melting curve analysis were used. The MTHFR C677T genotypes were determined in 189 patients with ESCC and 141 unrelated healthy controls. The results showed that the MTHFR C677T C/C, C/T and T/T genotype frequencies among healthy controls were 17.7%, 38.3%, and 44.0%, respectively. There was no significant difference in MTHFR T/T genotype frequency between ESCC patients (42.3%) and healthy controls (χ²=0.089, P＞0.05) whereas the C/T genotype frequency among ESCC patient (49.2%) was only slightly higher than that among healthy controls (χ²＝3.890, P＜0.05). However, the frequency of the C/C genotype among ESCC patients (8.5%) was significantly lower than that among healthy controls (17.7%) (χ²=6.37, P＜0.05). The C/C genotype significantly reduced the risk for developing ESCC compared to the combination of the C/T and T/T genotypes (OR=0.43, 95% CI=0.22～0.84). The reduced risk was more evident among smokers and patients with family history of upper gastrointestinal cancers. It can be concluded that the MTHFR C677T homozygous wild type may play a protective role in the ESCC development in the Northern Chinese population.