Low consumption of vegetables and fruits, which leads to insufficient folate intake, is associated with increased risk of several types of cancer, including head and neck squamous cell carcinoma (HNSCC). Functional polymorphisms in genes encoding one-carbon metabolism enzymes, such as methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G) and thymidylate synthase (TS), influence folate metabolism and thus might impact on HNSCC risk. We conducted a case-control study with 237 HNSCC cases newly and histologically diagnosed and 711 age- and sex-matched non-cancer controls to clarify associations with these five polymorphisms. Gene-environment interactions between polymorphisms and smoking and drinking habit and folate consumption were also evaluated by logistic regression analysis. Dietary folate intake was inversely associated with HNSCC risk. None of the polymorphisms showed any significant impact on HNSCC risk by genotype alone, but we found interactions between drinking habit and MTHFR C667T (P = 0.04), MTR A2756G (P = 0.04) and MTRR A66G (P = 0.03) polymorphisms. The results suggest that there may be interactions between one-carbon metabolism-related polymorphisms and alcohol drinking for HNSCC risk.