Polysaccharides are closely associated with immune regulation. In this study, the aim was to investigate the effect of polysaccharides from Cordyceps gunnii mycelia (PPS) in cyclophosphamide (CTX)-induced immunodeficient mice. Compared with the CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice with orally administered PPS were significantly increased, body weight loss was alleviated, and the natural killer (NK) cytotoxicity and proliferative activities of the lymphocytes were elevated. The recovery of peripheral white blood cells, red blood cells, hemoglobins and platelets was accelerated. Furthermore, the results from ELISA showed that PPS could up-regulate the serum levels of IL-2, IL-12, IFN-γ and IgG, and reduce the level of TGF-β. Histopathological analysis of the spleen revealed the protective effect of PPS against CTX-induced immunosuppression. Western blotting results showed that PPS possessed immunomodulatory activity via TLR4/TRAF6/NF-κB signalling pathways. Finally, the intestinal absorption of PPS was poor, as detected in the Caco-2 transwell system. Taken together, these findings suggest that PPS plays a crucial role in protection against immunosuppression in cyclophosphamide-treated mice and could be a potential candidate for use in immune therapy regimens.