The effect of homocysteine (Hcy) on extracellular-superoxide dismutase (EC-SOD) methylation levels in the aorta of mice was observed and the underlying mechanism of THP-1 monocyte-derived macrophages was investigated. The key targets of Hcy-induced EC-SOD methylation changes were identified. ApoE-/mice were fed different diets for 15 weeks and the concentrations of Hcy and lipids were subsequently detected using an automatic biochemistry analyzer. Serum levels of Hcy and lipids were significantly higher in the Meth group compared with the control group. EC-SOD and DNA methyltransferase (DNMT)1 mRNA and protein expression levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis, respectively. EC-SOD methylation levels were assessed using nested touchdown methylation-specific PCR (ntMS-PCR). Oxidative stress was measured using chromatometry. Recombined vectors overexpressing and silencing EC-SOD were transfected into macrophages. Following the transfection of macrophages with pEGFP-N1-DNMT1, the methylation levels of EC-SOD were detected. Furthermore, EC-SOD DNA methylation levels were significantly decreased in the Meth group. The promoter activity of EC-SOD was increased following transfection with-1/-1100 fragment and DNMT1 mRNA expression was significantly decreased in the Meth group. Following transfection with the recombinant plasmid, DNMT1 protein expression was significantly increased. ntMS-PCR results revealed that EC-SOD methylation levels significantly decreased in the Meth group, while they increased in N1-DNMT1 groups compared with the controls. In conclusion, Hcy may alter the DNA methylation status and DNMT1 acts as the essential enzyme in the methyl transfer process to disturb the status of EC-SOD DNA methylation, leads to a decrease in the expression of EC-SOD to promote oxidative stress and AS.