Abstract: This study aimed to investigate the effect of steam-exploded insoluble dietary fiber from Flammulina velutipes root (SE-IDF) on blood lipids and liver protection. The insoluble dietary fiber was prepared using an enzymatic method, and its chemical composition and fundamental physicochemical properties were characterized. A hyperlipidemia model was established in C57BL/6J male mice (n=40) induced by high-fat diet feeding. Mice were randomly divided into five groups: Normal diet group (ND), high-fat diet group (HFD), and three SE-IDF treatment groups with low (SE-IDFL, 50 mg/kg), medium (SE-IDFM, 100 mg/kg), and high doses (SE-IDFH, 200 mg/kg). After 10 weeks of intervention, serum and hepatic levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured. In addition, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lipopolysaccharide (LPS), and interleukin-6 (IL-6) were assessed. Hepatic oxidative stress markers, including total superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA), were also determined. Hepatic steatosis and lipid distribution were assessed by hematoxylin-eosin (HE) and oil red O staining to evaluate pathological changes in liver tissue. The results indicated that compared with IDF without steam explosion treatment, the oil holding capacity and cholesterol binding capacity of SE-IDF were obviously improved, resulting in good oil adsorption capacity. Animal experiments demonstrated that, compared with the HFD group, the SE-IDFH group significantly reduced serum and hepatic TC, TG, and LDL-C levels (P<0.05), and increased HDL-C levels (P<0.05). All SE-IDF treatment groups enhanced SOD and CAT activity as well as GSH levels, while decreasing MDA, LPS, and IL-6 concentrations, thereby alleviating inflammation and oxidative stress induced by a high-fat diet. SE-IDFM and SE-IDFH groups markedly improved liver histopathological structure, reduced hepatic lipid droplet area, vacuole formation, and inflammatory infiltration. These findings suggest that SE-IDF can effectively improve dyslipidemia in hyperlipidemic mice, reduce hepatic lipid accumulation, and exert hypolipidemic, hepatoprotective, and anti-fatty liver effects.