This study investigated the purification and characterization of a new immunomodulatory GlcNAc-containing polysaccharide (MIPB70-1) from Morchella importuna with molecular weights of 20.6 kDa. Structural analysis indicated that MIPB70-1 was composed of GlcNAc:Gal:Glc:Man with molar ratios of 1.00:7.16:5.54:5.61, and its primary structure was characterized as a repeating unit consisting of →6)-α-D-Glcp-(1→, α-D-GlcpNAc-(1→, α-D-Galp-(1→, β-D-Glcp-(1→, →6)-α-D-Manp-(1→, →4)-α-D-GlcpNAc-(1→, →4)-β-D-Glcp-(1→, →3,6)-α-D-Manp-(1→, →2)-α-D-Galp-(1→, →2,3,6)-α-D-Manp-(1→. Immunological assays indicated that MIPB70-1 enhanced the phagocytic function and promoted the secretion of nitric oxide (NO) as well as cytokines through targeting Toll-like receptor 4 (TLR4) on macrophage membrane and activating the downstream signaling pathways in RAW 264.7 cells. MIPB70-1 regulated mouse immunity to counteract the immune damage caused by the chemotherapy drug cyclophosphamide (CTX) in vivo. Furthermore, MIPB70-1 enhanced the anti-tumor activity of doxorubicin (DOX) and inhibited the growth of tumors, by immunomodulation in the orthotopic murine model of 4T1 breast cancer. These results demonstrate the potential of this GlcNAc-containing polysaccharide as an immune enhancer.