Sea buckthorn seed oil (SBSO) has been used as a functional food in the prevention of heart diseases. The present study investigates the effects of SBSO on blood cholesterol and the gut microbiota in hypercholesterolemia hamsters. Four groups of hamsters (n = 8 each) were given one of four diets, namely a non-cholesterol control diet (NCD), a high-cholesterol control diet (HCD) containing 0.1% cholesterol, and an HCD diet with sea buckthorn seed oil replacing 50% lard (SL) or replacing 100% lard (SH). Feeding SL and SH diets could reduce blood total cholesterol by 20–22%. This was accompanied by the down-regulation of the gene expression of acyl-CoA:cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP-binding cassette transporter8 (ABCG8). SBSO supplementation also increased the production of intestinal short-chain fatty acids and fecal outputs of neutral sterols. Metagenomic analysis demonstrated that feeding SL and SH diets could favorably modulate the relative abundance of Bacteroidales_S24-7_group, Ruminococcaceae, and Eubacteriaceae. It was therefore concluded that SBSO was effective in reducing blood cholesterol in hypercholesterolemic hamsters via increasing intestinal cholesterol excretion and promoting the growth of SCFA-producing bacteria.