Gut microbiota can help hosts maintain homeostasis, enhance nutrient absorption and energy metabolism, which is of great significance for the survival and environment adaption. Many factors can affect the gut microbiota, including microhabitat, captivity, seasonal changes, feeding items, sex, and development stage. In this study, the Big-eyed Ratsnake (Ptyas dhumnades) from Guilin and Xiangyang populations were selected as the research objects. The total microbial DNA was extracted from snake feces to sequence the V1﹣V3 regions of 16S rRNA gene using the amplicon sequencing and the differences in gut microbial composition, abundance, and gene function between the two populations were analyzed. At the phylum level, the dominant phyla included Bacteroidetes, Proteobacteria, Firmicutes and Fusobacteria (Fig 1). Linear discriminant analysis effect size (LefSe) analysis showed richly distributed bacteria and gene functions were different in the two populations (Fig. 2). However, there was no significant differences in a- diversity between the two populations (Fig. 3). The relatively high bacterial abundance in the Guilin population included the order Cardiobacteriales, the family Wohlfahrtiimonadaceae and the genus Koukoulia in Proteobacteria, and the members of the order Acidaminococcales and the genus Phascolarctobacterium in Firmicutes. However, the Xiangyang population owned the relatively high proportion in the order Burkholderiales, the family Aquaspirillaceaeke and the genus Microvirgula of Proteobacteria. In addition, the gene functional differences of gut microbiota between two populations were reflected in the high expression related to environmental information processing including ko02010 and ko02060 in Guilin populations. In contrast, those related to the ko02020 and ko00910 were mainly expressed in the Xiangyang population (Fig. 4). The high expression genes were related to the metabolism including ko00010, ko00520, ko00230 and ko00240 in the Guilin population, and ko00680 and ko00900 in the Xiangyang population (Fig. 4). Guilin population expressed a highly of ko03018, ko03010 and ko00970, while Xiangyang population expressed highly of ko02030. In the future, more studies are needed to focus on the composition of gut microbiota and the evolutionary relationships between gut microbiota and their hosts to provide relevant suggestions for the protection of wild animals.