Homocysteine (Hcy) is an independent risk factor for cardiovascular disease, but the molecular mechanisms causing atherosclerosis in monocytes remain poorly characterized. The objective of the present study was to investigate the effects of Hcy on DNA methylation of PPARα,γ and the underlying mechanism of PPARα,γ expression that was induced by Hcy in monocytes. About 50, 100, 200, and 500 μM Hcy were added to the monocytes cultured for 48 h. PPARα,γ that acted as lipid sensors and bind with mM affinities to ligands of antiatherosclerosis were determined by real-time reverse transcription–polymerase chain reaction and Western blotting in monocytes. Here, we used a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time polymerase chain reaction to determine the levels of the PPARα,γ DNA methylation. S-adenosylmethionine (SAM) level and S-adenosylhomocysteine (SAH) level were detected by high performance liquid chromatography. Results indicated that the levels of PPARα,γ promoter methylation in monocytes cultured with Hcy were increased in comparison with the control group, and the peak was in the 100 μM Hcy group, however, a dose-dependent increase with increasing Hcy was not seen. Hcy also decreased mRNA and protein levels of PPARα,γ in monocytes. Further, with the addition of Hcy, the levels of SAH were elevated, the levels of SAM and the ratio of SAM/SAH were lower, and the activity of C-5MT-ase was increased. In conclusion, these results suggest that PPARα,γ DNA methylation induced by Hcy may represent an important mechanism to explain atherosclerosis, which may become a therapeutic target for preventing atherosclerosis induced by Hcy.