Astragalus polysaccharide is one of the most extensively studied traditional Chinese medicinal polysaccharides because of its immunomodulatory activity and has attracted considerable attention. Existing evidence suggests that its potential immunomodulatory mechanism is related to the modulation of intestinal microbiota. However, current research methods on the gut microbiota mainly focus on 16S rRNA sequencing, providing limited evidence of specific changes in functional bacterial groups in the intestine. Butyrate is a class of short-chain fatty acids among the microbial metabolites in the gut and is most closely associated with immunomodulatory activity. Thus, in this study, we extracted and purified a polysaccharide from astragalus composed of a main chain of →4)-α-D-Glcp-(1 → and →4,6)-α-D-Glcp-(1→, with side chains of →6)-α-D-Glcp-(1→ and aggregated arabinose, and investigated the changes in butyrate-producing bacterial groups in mice during the immunomodulation process of astragalus polysaccharide, using two butyrate-producing bacterial-specific primers. The results showed that oral administration of astragalus polysaccharide significantly increased butyrate production in the mouse intestine, restoring the disrupted butyrate-producing bacterial abundance and diversity caused by immunosuppression. In conclusion, our study provides the first evidence of the targeted modulation of the butyrate-producing gut microbiota by astragalus polysaccharide, offering insights into its pharmacological activity.