Objective To explore the content and gene expression of endothelial nitric-oxide synthase (eNOS) in heart of hyperlipidemia apoE-/- mice. Methods apoE-/- mice were fed with high fat diet as high-fat diet group (n= 7) and normal fat diet as control group (n = 6) for 12 weeks. Serum lipid profiles including total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) were detected, eNOS mRNA expression in heart were determined by Real-Time RT-PCR, as well as protein expression by immunohistochemistry. Results Levels of serum TC, TG and LDL clearly increased in high fat diet group, while HDL decreased (P< 0. 01). mRNA and protein expression of eNOS in heart reduced in high fat diet group(P<0.01). Conclusions High fat diet may lead to hyperlipidemia in apoE-/- mice, which may down-regulate eNOS expression and decrease eNOS content in heart.