Aims The gut microbiota has a great effect on the health and nutrition of the host. Manipulation of the intestinal microbiota may improve animal health and growth performance. The objectives of our study were to characterize the faecal microbiota between wild and captive Tibetan wild asses and discuss the differences and their reasons. Methods and Results Through high-throughput sequencing of the 16S rRNA V4-V5 region, we studied the gut microbiota composition and structure of Tibetan wild asses in winter, and analysed the differences between wild and captive groups. The results showed that the most common bacterial phylum in Tibetan wild ass faeces samples was Bacteroidetes, while the phylum Firmicutes was dominant in captive Tibetan wild ass faecal samples. The relative abundance of Firmicutes, Tenericutes and Spirochaetes were significantly higher (P < 0 center dot 01) than in the wild groups. Conclusions Captivity reduces intestinal microbial diversity, evenness and operational taxonomic unit number due to the consumption of industrial food, therefore, increasing the risk of disease prevalence and affecting the health of wildlife. Significance and Impact of the Study We studied the effect of the captive environment on intestinal micro-organisms. This article provides a theoretical basis for the ex-situ conservation of wild animals in the future.