Objective To investigate the microbial composition of different intestinal segments of wild and captive tree shrews. Methods Seven samples of gastrointestinal segments of three healthy wild and captive tree shrews were collected to extract total bacterial DNA and adopt high-throughput sequencing technology. The V3-V4 hypervariable region of the 16SrRNA gene was sequenced, and the structure and diversity of the flora were analyzed. Results The diversity of the alpha index in different intestinal segments revealed significant differences between the wild and captive groups in the cecal and stomach segments (P< 0. 05). The microbial compositions of the intestines in the wild and captive groups were significantly different. At the gate level, the average proportion of Firmicutes in the rectum, colon, cecum, and stomach of the wild group was significantly higher than that of the captive group (P< 0. 05). In contrast, the average proportion of Spirochetes in the rectum, colon, cecum, and ileum of the captive group was significantly higher than that of the wild group (P< 0. 05). The average proportion of Bacteroidetes in the rectum was significantly higher in the wild group than in the captive group (P < 0. 05). At the genus level, the intestinal diversity of the wild group was significantly higher than the main genus of the captive group: the Weissella genus in the ileum, jejunum, and stomach; Bacteroides in the rectum; and Lactococcus in the ileum. The main breeding genus of the captive group was significantly higher than that of the wild group: Brachyspira in the rectum, colon, cecum, and ileum; Prevotella_9 in the cecum and stomach; Streptococcus in the cecum and jejunum; and Lactobacillus in the ileum. The functional gene prediction of the different intestinal segments revealed statistically significant differences among the 17 gene types in the wild and captive groups (P< 0. 05). Conclusions The intestinal flora composition in the intestines of wild and captive tree shrews is quite different. The bacterial function of each intestine is different, and is related to its physiological function and dietary structure. When studying intestinal flora, it is therefore necessary to consider whether the composition of the microbial sample microorganisms fully represents the composition of the gut microbes.