Intestinal microbiota play an important role in the survival of the host. However, no study to date has elucidated the adjustment of intestinal microbiota of the host during rewilding. Thus, this study aims to describe the intestinal bacterial community of reintroduced Przewalski’s horse (RPH) after being released into their original habitat for approximately 20 years in comparison with that of captive Przewalski’s horse (CPH), sympatric domestic horse (DH) and Mongolian wild ass (MWA) by sequencing the 16S rRNA gene. The results showed that the prevalent bacterial communities were different among CPHs, RPHs, DHs and MWAs at the family level. NMDS and ANOSIM analysis showed that the pattern of bacterial community composition in captive equines was distinct from that in the wild groups. It is shown that some bacteria had significant differences among different taxa (p < 0.001), such as Firmicutes, Bacteroidetes, Armatimonadetes, Clostrida, Bacteroidia, Clostridiales, Bacteroidales, Rikenellaceae and Bacteroidales_UCG-001. These bacteria were associated with the transition from in captive to in the wild (CPH and RPH), which reflected the change of environmental conditions. Meanwhile, Proteobacteria, Clostridia, Bacilli, Negativicutes, Gammaproteobacteria, Clostridiales, Bacillales, Selenomonadales, Pseudomonadales and Planococcaceae were the changed groups among RPHs, MWAs and DHs, which are related to feeding habits and diseases. Our results clearly showed the differences between intestinal microbiota in reintroduced animals and wild animals and led us to understand the survival state of reintroduced animals in the wild.