Polysaccharides are one of the primary active components of Radix Hedysari, although their regulatory effects on gut microbiota remain poorly understood. In this study, Radix Hedysari polysaccharides (RHPS) were isolated and purified, yielding three fractions: RHPS-1, RHPS-2, and RHPS-4. The yields of these fractions were 51.33 %, 3.15 %, and 2.34 %, respectively, with weight-average molecular weights of 18.781, 25.660, and 100.149 kDa. The three polysaccharides were composed of arabinose, galactose, glucose, glucuronic acid. RHPS-1 exhibits good antioxidant, antibacterial, and immune-enhancing activities. Further purification of RHPS-1 yielded RHPS-1-1, and it was found that RHPS-1-1 enhances the growth of beneficial bacteria while suppressing the growth of harmful bacteria in mice. Additionally, mice treated with RHPS-1-1 were primarily involved in bile acid, short-chain fatty acid, and energy metabolism pathways. Our results represent the first demonstration that RHPS-1-1 exhibits good biological activity and possesses the ability to regulate the gut microbiota and its metabolites in mice.