The purpose of this study was to observe the effect of Inonotus obliquus polysaccharide (IOP) on blood lipids and its regulation on the intestinal flora in hyperlipidemia rats, and explore the modern biological connotation of IOP in reducing blood lipids. In this study, we obtained the crude IOP by the water extraction and alcohol precipitation method, and then classified it by DEAE ion‐exchange chromatography to obtain the acidic I. obliquus polysaccharide (IOP‐A). After the administration of the IOP‐A, the serum TC, TG, and LDL‐C levels were significantly lower, while the serum HDL‐C levels were significantly higher. The expression of CYP7A1 protein was considerably increased, whereas the expression of SREBP‐1C protein was considerably decreased in the rat hepatic tissue. In addition, the IOP‐A could significantly alleviate the hepatocyte fatty degeneration in the liver lobule of rats. We believe that the IOP‐A can affect the composition of intestinal flora by reducing the relative abundance of Firmicutes and increasing the relative abundance of Bacteroidetes. These findings indicated that the IOP‐A can regulate the dyslipidemia of hyperlipidemia rats, and its mechanism may be through regulating the CYP7A1 and SREBP‐1C expression in the metabolism of lipids, and correcting the imbalance of intestinal flora structure caused by a high‐fat diet.