Abstract: Adopting a high-fat diet to induce a mouse model of hypercholesterolemia, the effect of Auricularia auricula aqueous extract (AE) on maintaining cholesterol health levels was evaluated by measuring body weight, organ index, serum, liver, and fecal biochemical parameters, pathological sections of epididymal fat and liver, and the mechanism of action was further investigated using widely targeted lipidomics. Compared to the control group (NFD), the serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels of the mice in the model group (HFD) were significantly increased, indicating the successful establishment of a hypercholesterolemia mouse model. Compared with the HFD group, epididymal fat index, serum TC, liver nonesterified fatty acid (NEFA) content, and liver index were significantly decreased by 32.05%, 13.94%, 75.00%, 16.41%, respectively, and the total content of short-chain fatty acids (SCFAs) were increased by 162.16%, and the hepatic steatosis and epididymal fat deposition were significantly ameliorated in the 500 mg·kg⁻¹ AE treated group (HAE). Thus, AE helped maintain cholesterol health levels. A total of 86 biomarkers were identified in the serum lipids of the HAE and HFD groups (P<0.05, VIP>1.0), of which 36 lipids such as phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), phosphatidylserine (PS) and lysophosphatidylserine (LPS) were up-regulated, and 50 lipids such as phosphatidylcholine (PC), ceramide (Cer), hexose ceramide (HexCer), hexose ceramide sulfide (SHexCer), phosphatidylinositol (PI), lysophosphatidylinositol (LPI) and phosphatidylglycerol (PG) were down-regulated, which mainly related to insulin resistance, AGE-RAGE signaling pathway in diabetic complications, adipocytokine signaling pathway, glycerolipid metabolism, cholesterol metabolism and other pathways. In summary, HAE significantly reduced serum TC, hepatic NEFA, liver index, and epididymal fat index, significantly increased fecal SCFAs, improved considerably liver steatosis and epididymal fat deposition, and thus helping maintain cholesterol health levels in high-fat diet-induced mice. The widely targeted lipidomics method revealed the mechanism of maintaining cholesterol healthy levels of HAE by regulating 86 lipid biomarkers in metabolic pathways such as insulin resistance, AGE-RAGE signaling pathway in diabetic complications, adipocytokine signaling pathway, glycerolipid metabolism, cholesterol metabolism.