Highland barley (Hordeum vulgare L. HB) has been demonstrated to have a series of dietotherapy values, including being low fat, low sugar, high fiber, and especially high in β-glucan. Long-term consumption could reduce the incidence of chronic diseases and metabolic syndromes. In this study, the regulating effect of modified highland barley (MHB) products, namely microwave fluidized HB, extruded and puffed HB, and ultrafine pulverized HB on lipid metabolism and liver injury in mice fed a high fat and cholesterol diet (HFCD) was investigated using microbiota diversity gene sequencing and untargeted metabolomics. A total of six groups of mice were supplemented with a normal diet or an HFCD, with or without MHB, and the experimental period lasted 10 weeks. The obtained results demonstrated that MHB supplementation could effectively reverse the increase in body weight gain and adipose tissue accumulation caused by an HFCD (p < 0.05). Moreover, serum biochemical parameters showed that MHB supplementation significantly decreased total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels, while increasing high-density lipoprotein cholesterol (HDL-C) levels. The results of hematoxylin and eosin (H&E) assays showed that MHB supplementation could significantly improve the liver injury and adipose tissue accumulation. In addition, 16S rRNA amplicon sequencing showed that MHB supplementation increased the bacteroidetes/firmicutes ratio and the abundance Lactobacillus abundance, while also decreasing the Proteobacteria abundance, which are bacteria closely associated with the hyperlipidemia caused by HFCD. LC-MS metabolomics indicated that MHB supplementation significantly enhanced the levels of Deoxycholic acid, Myclobutanil, 3-Epiecdysone, 3,4-Dihydroxybenzeneacetic acid, and so on. In addition, MHB supplementation promoted activation of the Arachidonic acid metabolism pathways, the expression of ABC transporters, bile secretion, primary bile acid biosynthesis, and so on. Above all, this study showed the potential capacity of MHB to relieve hyperlipidemia and provides a reference for developing a new dietary intervention supplement to ameliorate hyperlipidemia.