Simple SummaryWild Daurian ground squirrels (Spermophilus dauricus) breed only a few months out of the year, a behavior known as seasonal breeding. Despite the gut microbiota being an essential “organ” of animals, little is understood about how they relate to seasonal breeding. In the present investigation, metagenomic sequencing techniques were employed to examine the diversity of gut microbiota in wild Daurian ground squirrels across different breeding seasons. The findings indicate notable variations in the gut microbiota’s structure and function among wild Daurian ground squirrels during different seasons. This study may provide an in-depth discussion of how seasonal reproduction affects gut microbes and aid in analyzing how changes in gut microbes act on the host. This study could provide new insights into the seasonal reproductive behavior of animals as well as a new theoretical basis for the study of gut microbiology.AbstractTheSpermophilus dauricus,the wild Daurian ground squirrel, is known to exhibit seasonal breeding behavior. Although the importance of gut microbiota in animal digestion, metabolism, and immunity is well-established, the correlation between gut microbiota and seasonal breeding in this species remains inadequately explored. In the present study, using metagenomic sequencing technology, the compositions and functions of the gut microbiota of wild Daurian ground squirrels in different breeding seasons were explored. The dominant gut microbial phyla were Firmicutes and Bacteroidetes. The Firmicutes were predominant in the breeding season, whereas Bacteroidetes were predominant in the non-breeding season. At the genus level,Lactobacillusaccumulated during the breeding season, whereasOdoribacterandAlistipesincreased during the non-breeding season. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genome) annotations indicated that genes in gut samples were highly associated with metabolic functions. The differential expression gene analysis showed that genes related to the phosphotransferase system, cysteine, and methionine metabolism were highly expressed during the breeding season, whereas the non-breeding season upregulated genes were enriched in starch and sucrose metabolism and bacterial chemotaxis pathways. In conclusion, this study could provide a reference for investigating gut microbiota in seasonal breeding animals and offer new insight into gut microbial function.