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The molecular mechanism underlying the chemoprotective effects of Ganoderma atrum polysaccharide in cyclophosphamide-induced immunosuppressed mice was investigated. In Cy-treated mice, PSG-1 treatment significantly promoted the phagocytosis, and stimulated the production of NO and cytokines (TNF-α and IL-1β) in peritoneal macrophages. Moreover, PSG-1 elevated the phosphorylation of MAPKs and Akt, as well as expression of NF-κB in peritoneal macrophages. In addition, PSG-1 enhanced the recovery of T and B cell proliferation responses in Cy-treated mice. Furthermore, Ca2+ concentration and PKC activity of spleen lymphocytes in PSG-1 groups dramatically increased as compared with that of the model group. Finally, PSG-1 administration was found to dose-dependently improve the decline of cAMP level and PKA activity caused by Cy. These findings indicated that the chemoprotective effects of PSG-1 may be attributed to its capacity to activate peritoneal macrophages and spleen lymphocytes in Cy-treated mice.