A novel bioactive polysaccharide, GFP-22, was isolated from the fruit bodies of Grifola frondosa by anion-exchange and gel filtration chromatography. Structure of GFP-22 was investigated using gas chromatography–mass spectrometry (GC–MS), methylation, nuclear magnetic resonance (NMR), high-performance size exclusion chromatography-multi-angle laser light scattering-refractive index detector (HPSEC-MALLS-RI) and atomic force microscopy (AFM) analysis. The backbone of GFP-22 is composed of 1,4-β-D-Glcp, 1,3-β-D-Glcp, 1,6-α-D-Glcp, 1,6-α-D-Galp, 1,4,6-α-D-Manp and 1,3,6-α-D-Manp units. Molecular weight of GFP-22 is 2.72 × 104 Da. GFP-22 has a linear filamentous structure. The administration of GFP-22 could improve or reverse the CTX-induced immunosuppression, significantly enhance the spleen and thymus indices, spleen lymphocyte proliferation and cytokines production in splenocytes. These findings suggest that GFP-22 could be explored as a natural and potential immunomodulatory agent and as an alternative means of lessening chemotherapy-induced immunosuppression.