Reintroduction is an effective strategy in the conservation of endangered species under scientific monitoring. Intestinal flora plays an important role in the envir onmental adaptation of endangered Père David’s deer (Elaphurus davidianus). In this study, 34 fecal samples from E. davidianus were collected from different habitats in Tianjin city of China to investigate differences in the intestinal flora under captive and semi-free-ranging conditions. Based on 16S rRNA high-throughput sequencing technology, a total of 23 phyla and 518 genera were obtained. Firmicutes was dominant in all individuals. At the genus level, UCG-005 (13.05%) and Rikenellaceae_RC9_gut_group (8.94%) were dominant in captive individuals, while Psychrobacillus (26.53%) and Pseudomonas (11.33%) were dominant in semi-free-ranging individuals. Alpha diversity results showed that the intestinal flora richness and diversity were significantly (P < 0.001) higher in captive individuals than in semi-free-ranging individuals. Beta diversity analysis also showed a significant difference (P = 0.001) between the two groups. In addition, some age- and sex-related genera such as Monoglobus were identified. In summary, the structure and diversity of intestinal flora showed significant habitat variation. This is the first time an analysis has been undertaken of the structural differences of the intestinal flora in Père David’s deer, under different habitats in the warm temperate zone, providing a reference basis for the conservation of endangered species.