Aim To investigate the mechanism of endothelial nitric oxide synthase （eNOS） promoter DNA methylation in the damage of endothelial cells induced by homocysteine （Hcy）. Methods Human umbilical vein endothelial cells （HUVEC） were cultured with Hcy at different concentrations （0,50,100,200 and 500 μmol/L） and 100 μmol/L Hcy+Vitamin B12+folate respectively for 72 hours. The cell viability of HUVEC was detected by MTT,the mRNA expression of eNOS was analyzed by real-time PCR. The activity of eNOS was analyzed by ELISA,and the production of nitric oxide （NO） was examined by a commercial kit. Nested methylation specific polymerase chain reaction （nMS-PCR） was used for analysis of the methylation pattern in promoter regions of eNOS gene. The DNA methyltransferase activity was measured by isotope method. Results The viability of HUVEC exposed to different concentrations of Hcy decreased significantly. The mRNA levels of eNOS,eNOS activity and the NO production were significantly decreased. The methylation levels of eNOS promoter were Hcy-dose dependently increased,and the DNA methyltransferase activity was correlative increased. Conclusion Homocysteine can induce DNA hypermethylation of eNOS promoter,and the eNOS gene expression displayed correlative decrease,which may be an important mechanism for homocysteine-mediated endothelial impairment.