The digestive tract of ruminants is the home of the gut microbiome ecosystem, which plays a huge role in the diagnosis of various health conditions and the analysis of physiological conditions in wild animals. Red deer is a second-class protected animal in China. In this study, we used microsatellite and high-throughput sequencing of the 16S rRNA gene in fecal samples of red deer to investigate differences in the gut bacterial microbiota were analyzed between wild and captive in winter. Our results revealed that proportions of bacterial taxa, alpha-and beta-diversities, and relative abundances of amplicon sequence variants in the gut bacterial microbiota of the two groups differed. Firmicutes (79.46%), Bacteroidetes (16%) and Tenericutes (1.25%) were the most predominant phyla in wild red deer. While in captive red deer, Firmicutes (62.5%) was the dominant phylum, followed by Bacteroidetes (29.1%) and Tenericutes.( 3.21%). The specific function and mechanism of Tenericutes in red deer need further study. The wild red deer had higher fecal bacterial diversity than the captive in farm. These differences were attributed to the enrichment of bacterial taxa involved in the digestion of the supplementary food and to different natural diets consumed in the forest. Also the dominant and differential microflora of intestinal microflora in various populations were mined and their related metabolic pathways. In terms of functional data, most of the genes annotated are related to metabolism. The second most commented gene is related to genetic information processing. The comparative study of the intestinal flora of the two populations can not only assess the health status of the two populations, but also provide important suggestions for the breeding of captive red deer and the protection of wild populations.