Both betaine homocysteine methyltransferase (BHMT) and cystathionine β-synthase (CBS) are major enzymes in the metabolism of plasma homocysteine (Hcy). Abnormal methylation levels of BHMT and CBS are positively associated with Hcy levels. The present study is performed to explore the association between the methylation levels in the promoter regions of the BHMT and CBS genes and the efficacy of folic acid therapy in patient with hyperhomocysteinemia (HHcy). A prospective cohort study recruiting HHcy (Hcy ≥ 15 μmol/L) patients was performed. The subjects were treated with oral folic acid (5 mg/d) for 90 days, and the patients were divided into the success group (Hcy < 15 μmol/L) and the failure group (Hcy ≥ 15 μmol/L) according to their Hcy levels after treatment. In the logistic regression model with adjusted covariates, the patients with lower total methylation levels in the BHMT and CBS promoter regions exhibited 1.627-fold and 1.671-fold increased risk of treatment failure compared with higher methylation individuals, respectively. Similarly, subjects who had lower methylation levels (<methylation mean) in BHMT CpG1 had 1.792 times higher risks. Stratified analysis by sex found that lower CBS methylation levels were associated with a 2.128-fold increased risk for treatment failure in males with HHcy. Lower levels of BHMT or CBS promoter total methylation might be associated with increased the risk of treatment failure. These studies suggest that lower levels of BHMT and CBS methylation are all predictors of failure in folic acid therapy for HHcy. However, due to some limitations of this study, such as the small number of the loci tested, further large-scale studies are necessary to verify our observations.