<b><i>Background/Aims:</i></b> Low folate intake may increase risk of colorectal cancer by altering gene-specific methylation in the colon. We determined whether supplementation with physiological doses of folate could alter methylation in the oestrogen receptor 1 (ESR1) and mutL homolog 1 (MLH1) genes in colonic mucosa of subjects with colorectal adenoma. <b><i>Methods:</i></b> This was a randomised, double-blind, placebo-controlled trial. Subjects received either 400 µg/day folic acid (n = 15) or placebo (n = 14) for 10 weeks. Blood and colonic tissue samples were collected at baseline and after intervention to determine biomarkers of folate and vitamin B₁₂ status, MTHFR C677T and MS A2756G genotypes, and ESR1 and MLH1 methylation. <b><i>Results:</i></b> Although serum and red cell folate increased (p < 0.001 vs. placebo) and plasma homocysteine decreased (p = 0018 vs. placebo) in the folic acid group, there were no significant changes in ESR1 (p = 0.649 vs. placebo) or MLH1 (p = 0.211 vs. placebo) methylation. There was a significant effect of gender on ESR1 methylation (p = 0.004) and significant gender and genotype (MTHFR C677T and MS A2756G) interactions (p = 0.04 and p = 0.014, respectively) that were independent of treatment group allocation. <b><i>Conclusions:</i></b> Short-term folate supplementation in physiological doses decreases plasma homocysteine but has no effect on ESR1 and MLH1 methylation in colonic mucosa of individuals with adenoma. Further studies to investigate the interactions between gender, genotype and DNA methylation suggested in this study are warranted.