Hyperlipidemia is a pathological disorder of lipid metabolism that can cause fatty liver, atherosclerosis, acute myocardial infarction, and other diseases, seriously endangering people's health. Polysaccharides have been shown to have lipid-lowering potential. In the current study, the anti-hyperlipidemia effect and potential mechanisms of a polysaccharide (BGP-Z31) obtained from barley grass harvested at the stem elongation stage in high-fat diet (HFD)-treated mice were investigated. Results showed that supplementation with BGP-Z31 (200 and 400 mg kg-1) not only suppressed obesity, organ enlargement, and fat accumulation caused by HFD, but also regulated dyslipidemia, relieved liver function injury, and ameliorated the oxidative stress level. Meanwhile, BGP-Z31 increased the concentrations of acetic acid, propionic acid, butyric acid, and isovaleric acid in HFD-induced mice. Gut microbiota analysis demonstrated that BGP-Z31 had no obvious effect on the gut microbiota diversity in mice treated with HFD, but it positively remodeled the intestinal flora structure by elevating the relative abundances of Bacteroides, Muribaculaceae, and Lachnospiraceae and lowering the Firmicutes/Bacteroides value and the relative abundance of Desulfovibrionaceae. Therefore, our data suggested that BGP-Z31 can be used as a promising nutritional supplement for dietary intervention in hyperlipidemia.