To investigate the effect of astragalus polysaccharide (APS) in the treatment of ulcerative colitis (UC) and its molecular mechanisms, particularly through the ability to regulate Treg cell-glycolytic interactions. BALB/c mice were used to establish DSS-induced UC model, and administered with APS (200 mg/kg) as a therapeutic intervention. During the study, flow cytometry was employed to analyze the changes in the proportions of Treg cells and their related subpopulations in the spleens of mice. Additionally, the expression levels of various cytokines in the colon tissues were detected simultaneously. To further investigate the mechanism of action of APS, RNA sequencing was also performed on the colon tissues, and the regulatory effects of APS on glycolysis and mTOR/HIF-1α signaling pathway were revealed by KEGG pathway analysis, western blot, RT-qPCR and other techniques. In this study, we found that APS not only substantially ameliorated the adverse symptoms in UC mice, but also promoted the expression of anti-inflammatory cytokines and suppressed the levels of pro-inflammatory cytokines by regulating the distribution of Treg cells and their subpopulations. Furthermore, the molecular mechanism by which APS exerts its protective effects by inhibiting glycolysis and mTOR/HIF-1α signaling pathway was revealed through RNA sequencing, KEGG pathway analysis, and other techniques. The study revealed that APS effectively ameliorated the symptoms of UC in mice by regulating Treg cells, inhibiting glycolysis and mTOR/HIF-1α signaling pathway, providing a scientific basis and potential therapeutic target for the application of APS in UC treatment.