Inactivation of the p16INK4A (CDKN2A) gene in the Rb pathway is among the most common somatic alterations observed in tobacco-related solid tumors, including head and neck squamous cell carcinoma (HNSCC). In addition, a low folate diet is an important risk factor for HNSCC. Decreased dietary folate in an animal model of hepatocellular carcinoma has been associated with the induction of epigenetic silencing of the p16INK4A gene. In an ongoing population-based study of HNSCC, we sought to extend this observation to human disease testing the hypothesis that p16INK4A methylation is associated with decreased dietary folate. We also investigated the association of methylation silencing with functional polymorphisms in the folate metabolism enzyme methylene tetrahydrofolate reductase (MTHFR). In 169 HNSCCs, the odds ratio for p16INK4A methylation among those with low dietary folate intake was 2.3 (95% CI = 1.1-4.8) when compared with those with high folate intake. Furthermore, this increased risk for epigenetic silencing at p16 INK4A was modified by the MTHFR alleles previously associated with diminished serum folate levels. Hence, in HNSCC low dietary intake of folate is associated with p16INK4A methylation, and this relationship is modified by the MTHFR genotype. Our data provides important evidence for a mechanism of action of folate deficiency in cancer.