Our study suggested that: firstly, healthy controls were more likely to have higher levels of vitamin B2 and B12 than ESCC cases; secondly, variant genotype TT and the T allele were associated with significantly increased risk of EPL; thirdly, the MTHFR C677T genotype may modify association between serum vitamin B2 or B12 levels and the risks of EPL and ESCC; fourthly, gene–gene interaction was observed as there were strong association between the interaction of p16 and p53 methylations and MTHFR C677T polymorphism and the risks of EPL and ESCC. This evidence concerns the gene MTHFR and esophageal squamous cell carcinoma.