Gut microbiota is closely related to animal nutrition metabolism, body health, immunity and other aspects, and can also indirectly reflect the environmental adaptability of species. In 2017, the footprint of the water deer (Hydropotes inermis) reappeared in the northeast China. The reasons for the population diffusion and recovery were preliminary explored. To effectively expand the distribution of water deer in China, fresh fecal samples of water deer were collected from four areas in southeast Liaoning Province in winter of 2021 by non-invasive sampling method. The V3-V4 hypervariable region of bacterial 16 Sr RNA in fecal DNA was amplified by high-throughput sequencing technology to analyze the composition and diversity of the gut microbiota of water deer. The results showed that Firmicutes and Bacteroidetes were the dominant phylum in the intestine of the water deer. Proteobacteria (30.40%) and Escherichia_Shigella (28.48%) were relatively abundant only in Shajianzi (SJZ), but less than 2% and 0.1% in the other three regions. There was no difference in the abundance of gut microorganisms in the four regions (ACE and Chao1 index, P ＞ 0.05), but there was significant difference in the diversity of gut microbiota between Shajianzi area and the other three areas (Shannon and Simpson, P ＜ 0.05). The results of NMDS analysis and ANOSIM analysis indicated that the structure of gut microbiota in Shajianzi area was similar to other three areas, but there were some differences among the groups (R ＞ 0, P ＜ 0). LDA histogram indicated that there were 18 species of bacteria in the intestine of water deer from the four areas. Anaerobutyricum was significantly enriched in the intestines of the water deer in the Xialuhe (XLH), while Peribacillus was the most influential species in Shajianzi. Through the analysis of the intestinal flora of the water deer distributed in the northeast region, the adaptation mechanism of the gut microbiota of the water deer living in the cold region was preliminary understood, and the potential pathogenic bacteria in the water deer and the way of obtaining energy by digesting crude fiber were revealed, which provided basic data for further exploring the physiological and ecological adaptation of the intestinal microorganisms of the water deer in Northeast China. It is suggested that some measures should be taken to protect or restore the food source of the water deer in the southeastern Liaoning Province, and to improve the biodiversity level in the suitable habitat of the water deer, so as to effectively protect the population to a certain extent.