The chemical structure, chain conformation, and immuno-modulatory activity of polysaccharide from mycelium Cordyceps sinensis fungus UM01 were investigated. The molecular weights and the intrinsic viscosity of purified polysaccharide (UM01-S4) were determined as 22,559 Da and 5.09 mL/g, respectively. Using the polymer solution theory, UM01-S4 has a sphere-like conformation in aqueous NaCl at 35 °C. The chemical structure of UM01-S4 contains an α-(1→2)-Manp core. The side chains were composed of β-(1→2)-Galf, β-(1→4)-Glcp, α-GalpA, and α-Manp units, which attached to the mannan core at the O-6 position. The immuno-modulatory assays showed that UM01-S4 exhibited good activities, including macrophages proliferation, phagocytosis, and release of NO and cytokines. The mechanism underlying the macrophage regulation of UM01-S4 might be related to the activation of the MAPK and NF-κB signalling pathways. Thus, mycelium of C. sinensis UM01 could be used as a natural immune-stimulating agent in functional food areas and pharmaceutical industries.