Hyperlipidemia constitutes a critical factor in the pathogenesis of vascular disease and gut microbiota dysbiosis. This study aims to investigate the role of sea buckthorn fruit oil (SBFO) in ameliorating hyperlipidemia, modulating gut microbiota, and elucidating the underlying mechanisms of its lipid-lowering effects. SBFO intervention in hyperlipidemic mice significantly reduced body weight and visceral adiposity index while mitigating symptoms associated with hyperlipidemia, including dyslipidemia, inflammation, liver damage, and endothelial dysfunction. Real-time quantitative PCR and Western blot analyses revealed that SBFO enhanced the response of free fatty acid oxidation and breakdown (adenosine 5’-monophosphate-activated protein kinase α, carnitine palmitoyltransferase1a, peroxisome proliferator-activated receptor α, and peroxisome proliferator-activated receptor γ) while suppressing the synthesis of triglycerides and fatty acids (sterol-regulatory element binding protein 1, fatty acid synthase, and acetyl CoA carboxylase) at the gene and protein expression levels. Moreover, SBFO intervention enriched the gut microbiota of hyperlipidemic mice by increasing the relative abundances of Lactobacillus, Faecalibaculum, and Allobaculum, while decreasing the Firmicutes/Bacteroidetes ratio. These findings suggest that SBFO exhibits promising therapeutic potential in attenuating hyperlipidemia in mice through the regulation of lipid metabolism and modulation of gut microbiota.