Abstract: Objective: To investigate the mechanism of Osmanthus fragrans ethanol extract (OFE) in ameliorating dyslipidemia via modulation of gut microbiota dysbiosis and enhancement of reverse cholesterol transport (RCT) in hyperlipidemic mice. Methods: Hyperlipidemic mice models were established by feeding a high-fat diet for 6 weeks, they were divided into five groups: normal control, model, OFE low-dose, OFE high-dose, and simvastatin groups. After 5 weeks of oral administration, body weight, fasting blood glucose, and liver index were measured. Serum lipid and oxidative stress levels, key proteins levels in the RCT pathway were measured. Hepatic histopathological changes were observed by HE staining, and gut microbiota diversity and compositional changes in feces were analyzed using 16S rRNA high-throughput sequencing. Results: OFE could reduced liver index and blood glucose levels in model mice (P<0.01); decreased serum levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels (P<0.05 or P<0.001), while elevating high-density lipoprotein cholesterol (HDL-C) levels (P<0.01 or P<0.001); reduced malondialdehyde (MDA) levels (P<0.01) and enhanced superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) activities (P<0.05, P<0.01, or P<0.001). Additionally, OFE ameliorated hepatic steatosis, suppressed inflammation, and mitigated cellular damage. It upregulated serum levels of ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein A1 (ApoA-Ⅰ) (P<0.001) and modulated gut microbiota diversity while reducing the Firmicutes to Bacteroidetes (F/B) ratio (P<0.05). Conclusion: OFE exhibited potential therapeutic effects in hyperlipidemic mice by attenuating lipid accumulation and oxidative stress levels, as well as improving dyslipidemia, which may be associated with its ability to enhance cholesterol reverse transport (RCT) and ameliorate gut microbiota dysbiosis.