To investigate the effects of orlistat on high-fat diet-induced obese rats, 30 rats were randomly grouped into normal group of 10 rats and high-fat diet group of 20 rats, and obese rats were obtained after 10 weeks. Then, these obese rats were randomly and evenly divided into model group(10 rats) and orlistat group(10 rats). The normal group, model group and orlistat group were given normal diet, high-fat diet, high-fat diet + orlistat intragastric administration, respectively. The serum samples were collected after 10 weeks of the experiment for serum biochemical index detection, histopathological observation and gut microbiota analysis. The results showed that orlistat significantly reduced the body mass of rats on a high-fat diet, while reducing the accumulation of lipids in the liver caused by a long-term high-fat diet and improving liver injury and the accumulation of body fat. Orlistat also reduced serum TC and LDL-C levels and increased HDL-C levels in rats on a high-fat diet, improving the dyslipidemia in obese rats. And orlistat treatment improved glucose insulin resistance by increasing serum lipocalin levels and decreasing serum leptin and free fatty acid levels in obese rats. In addition, orlistat intervention caused a reduction in the abundance and diversity of the small intestinal and cecum flora and an increase in the species diversity of the microbial community in the colon of rats. Orlistat reduced the ratio of the relative abundance of the Firmicutes to the Bacteroidetes in the colon of rats on a high-fat diet. The relative abundance of Spirochaetes and Verrucomicrobia in the cecum of rats after high-fat diet was significantly higher than that in the normal group, but the relative abundance of Spirochaetes decreased after orlistat treatment compared to the model group. Orlistat promoted the growth of beneficial intestinal bacteria, such as Lactobacillus and Romboutsia in the small intestine; Akkermansia and Blautia in the cecum; and Blautia, Lachnobacterium, Roseburia in the colon; and inhibited the colonization of harmful bacteria, such as Streptococcus in the small intestine. Therefore, the results of this study suggested that orlistat could effectively improve obesity and metabolic disorders caused by high-fat diet, and could improve the regulation of dysbiosis of gut microbiota caused by high-fat diet.