Hyperlipidemia is currently rising at an alarming rate in human populations around the world, and new innovative ways of alleviating hyperlipidemia are needed. The effects of Jerusalem artichoke inulin on hyperlipidemia and the intestinal microflora in mice fed high-fat diet (HFD) were investigated. Inulin-treated HFD-fed hyperlipidemic mice had decreased serum lipid hepatic triglyceride (TG), hepatic total cholesterol (TC) concentrations and atherogenic index (AI). The inulin treatment increased hepatic superoxide dismutase (SOD) activity and decreased hepatic malondialdehyde (MDA) concentration. Histological analysis of liver revealed a decrease in abundance of lipid droplets in inulin-treated HFD-fed hyperlipidemic mice. Illumina high-throughput sequencing technology was used to analyze the bacterial community structure of the intestinal contents of mice. Analysis showed that the inulin treatment improved intestinal microflora; in particular, it significantly increased the number of Bifidobacterium in the intestine of HFD-fed mice. Inulin is therefore potentially potent functional food for preventing and treating hyperlipidemia.