The protective effect of Lyophyllum ulmarium fibrinolytic enzyme (LUFE) on vascular endothelium injury in hyperlipidemic rats was investigated. The SD rats were randomly divided into normal control group, model group, positive control group, and LUFE group. The normal control group was fed with normal diet, while the rest groups were given high-fat diet, in which the LUFE group was simultaneously gavaged with LUFE (400mg·kg-1·d-1). Positive control group started gavage of atorvastatin calcium tablets (5mg·kg-1·d-1) at the 5 week of feeding. After 4 weeks and 8 weeks of rearing, the corresponding indexes were tested respectively. Hematoxylin-eosin staining was used to observe the pathological changes of aorta. Oil red O staining was used to observe the distribution of lipid plaques in aortic intima. The levels of plasma total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), catalase (CAT),malondialdehyde (MDA), superoxide dismutase 2 (SOD2), endothelin-1 (ET-1) and 6-keta-PGF1α were measured and the atherogenic index was calculated. Nuclear factor erythroid 2-related factor (Nrf2), silent information regulator 3(Sirt3), acetyl-superoxide dismutase 2(Ac-SOD2), and heme oxygenase 1(HO-1)of aorta were detected by Western blot. LUFE attenuated the pathological injury of aorta in hyperlipidemic rats, reduced the relative area of lipid plaque distribution in aortic intima, decreased the levels of TC, TG, LDL-C, ET-1 and MDA in plasma, and increased the levels of 6-keta-PGF1α, CAT and SOD2. The expression of Nrf2, HO-1 and Sirt3 protein in aortic tissue was up-regulated, and relative protein expression of Ac-SOD2 in aortic tissues was reduced. LUFE attenuated vascular endothelium injury induced by hyperlipidemia in rats, and the mechanism may be related to the reduction of blood lipid and oxidative stress injury.