Potato resistant starch (RS) was prepared by microwave-toughening treatment (MTT). This study investigated the beneficial effects of RS on high-fat diet (HFD)-induced hyperlipidemia in C57BL/6J mice by evaluating changes in the gut microbiota. The mice were fed low-fat diet with corn starch, HFD with corn starch, HFD with potato starch (HFP), or HFD with RS (HFR) for 6 weeks. The results showed that the HFR group had lower body weight and total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels compared with the HFP group. Moreover, the brown adipose tissue levels of uncoupling protein 1 (UCP-1), β3-adrenoceptor (β3-AR), peroxisome proliferator-activated receptor-γ (PPAR-γ), and PPAR-γ coactivator-1α (PGC-1α) were increased. Our results showed that RS supplementation increased the Bacteroidetes/Firmicutes ratio and the abundance of short-chain fatty acid-producing Allobaculum, Ruminococcus, and Blautia. Our data suggest that RS prepared by MTT may be used as a prebiotic agent to prevent gut dysbiosis and obesity-related chronic diseases, such as hyperlipidemia, and obesity.