Polysaccharide (PF40) has been recognized as a main bioactive substances inPseudostellaria heterophylla (Miq.). The current study explored the potential protective effects of PF40 on immune system in mice with cyclophosphamide-induced immunosuppression. The mice were intragastric administered PF40 at the dosage of 100, 200 or 400 mg/kg once daily for 30 days, On the 24th and 25th day, the additional intraperitoneal injection of PF40 (50 mg/kg) were administered. The results showed that PF40 enhanced the cell-mediated immunity via improvements in macrophage phagocytosis, splenocyte proliferation, NK cell activity and delayed type hypersensitivity. Equally, it improves humoral immunity through promoting the formation of serum hemolysin.Moreover, PF40 maintain the immune balance of splenic lymphocytes and altered the intestinal physiological status in Cyp-induced mice. PF40 regulates the intestinal microbiota by restoring the relative abundance of Odoribacter and Mucispirillum and reducing the relative abundance of Sporosarcina, Yaniella, and Jeotgalicoccus in Cypintervened mice. The findings suggested that PF40 might be a promising natural functional foods for reducing chemotherapy-induced immunosuppression.