The gut microbiota plays a crucial role in the host’s metabolic processes. Many studies have shown significant changes in the gut microbiota of mammals during hibernation to adapt to the changes in the external environment, but there is limited research on the colonic epithelial tissue and gut microbiota of the wild chipmunks during hibernation. This study analyzed the diversity, composition, and function of the gut microbiota of the wild chipmunk during hibernation using 16S rRNA gene high-throughput sequencing technology, and further conducted histological analysis of the colon. Histological analysis of the colon showed an increase in goblet cells in the hibernation group, which was an adaptive change to long-term fasting during hibernation. The dominant gut microbial phyla were Bacteroidetes, Firmicutes, and Proteobacteria, and the relative abundance of them changed significantly. The analysis of gut microbiota structural differences indicated that the relative abundance of Helicobacter typhlonius and Mucispirillum schaedleri increased significantly, while unclassified Prevotella-9, unclassified Prevotellaceae-UCG-001, unclassified Prevotellaceae-UCG-003 and other species of Prevotella decreased significantly at the species level. Alpha diversity analysis showed that hibernation increased the diversity and richness of the gut microbiota. Beta diversity analysis revealed significant differences in gut microbiota diversity between the hibernation group and the control group. PICRUSt2 functional prediction analysis of the gut microbiota showed that 15 pathways, such as lipid metabolism, xenobiotics biodegradation and metabolism, amino acid metabolism, environmental adaptation, and neurodegenerative diseases, were significantly enriched in the hibernation group, while 12 pathways, including carbohydrate metabolism, replication and repair, translation, and transcription, were significantly enriched in the control group. It can be seen that during hibernation, the gut microbiota of the wild chipmunk changes towards taxa that are beneficial for reducing carbohydrate consumption, increasing fat consumption, and adapting more strongly to environmental changes in order to better provide energy for the body and ensure normal life activities during hibernation.