Abstract Methylation of the promoter region of tumor suppressor genes is increasingly recognized to play a role in cancer development through silencing of gene transcription. We examined the associations between dietary folate intake, MTHFR C677T genotype, and promoter methylation of six tumor suppressor and DNA repair genes. Patients with colorectal adenoma (n = 149) and controls (n = 286) with folate intake in the upper or lower tertile with the CC or TT genotype were selected from a case-control study. Methylation-specific PCRs were conducted on colorectal adenoma specimens. The percentages of promoter methylation ranged from 15.7% to 64.2%. In case-case comparisons, folate was inversely associated with promoter methylation, especially among TT homozygotes. Case-control comparisons suggested that folate was not associated with the occurrence of adenomas with promoter methylation, and increased the risk of unmethylated adenomas, especially in TT homozygotes. The interactions between folate and MTHFR genotype were most pronounced for O6-MGMT: compared with CC homozygotes with low folate intake, the adjusted odds ratios (95% confidence interval) of having a methylated O6-MGMT promoter were 3.39 (0.82-13.93) for TT homozygotes with low folate intake and 0.37 (0.11-1.29) for TT homozygotes with high folate intake (P interaction = 0.02); the odds ratios for the occurrence of adenomas without methylation were 0.57 (0.16-2.11) for TT homozygotes with low folate intake and 3.37 (1.17-9.68) for TT homozygotes with high folate intake (P interaction = 0.03). In conclusion, folate intake seems to be inversely associated with promoter methylation in colorectal adenomas in case-case comparisons, and was positively associated with the occurrence of adenomas without promoter methylation in case-control comparisons, especially for TT homozygotes. (Cancer Epidemiol Biomarkers Prev 2007;16(2):327–33)