Di (2-ethylhexyl) phthalate (DEHP) and high-fat diet (HFD) could induce lipid metabolic disorder. This study was undertaken to identify the effect of DNA methylation of JAK3/STAT5/PPARγ on lipid metabolic disorder induced by DEHP and HFD. Wistar rats were divided into a normal diet (ND) group and HFD group. Each diet group treated with DEHP (0, 5, 50, 500mg/kg/d) for 8weeks' gavage. The DNA-methylated levels of PPARγ, JAK3, STAT5a, and STAT5b in rats' livers and adipose were analyzed with MethylTarget. The lipid levels of rats' livers and adipose were detected with ELISA. Results showed in ND group that the DNA methylation levels of PPARγ, JAK3 in livers, and STAT5b in adipose were lower in 500mg/kg/d group than the control. And the level of total cholesterol (TC) in adipose was higher in 500mg/kg/d group than the control. In HFD group, the DNA methylation level of JAK3 was the lowest in livers and the highest in adipose in 50mg/kg/d group. And the level of TC in livers was the lowest in 50mg/kg/d group. In the 500mg/kg/d group, the DNA methylation level of STAT5b was lower in livers and higher in adipose in HFD group than that in ND group. And the levels of TC in livers were lower in HFD group than those in ND group. Therefore, DNA methylation of JAK3/STAT5/PPARγ regulated the changes in lipid levels induced by DEHP and HFD in adolescent rats.