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 the contents of cellulose, hemicellulose, and lignin in Flammulina velutipes root were significantly reduced by steam explosion treatment. 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.