β-Glucans derived from various sources such as yeast cell walls and medicinal mushrooms are considered as valuable biological response modifiers for their ability to enhance the activity of immune cells, aid in wound healing and help prevent infections. We herein characterize the structure of a novel water-soluble polysaccharide ( Zhuling polysaccharide, ZPS) from the fruit bodies of medicinal mushroom Polyporus umbellatus and investigate its immunobiological function. ZPS has a molecular mass of 2.27 10 3 kDa and contains 〉90% d -glucose as its monosaccharide constituent. On the basis of partial acid hydrolysis, methylation analysis, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy and the ideal repeating unit of ZPS is established: (1 -〉 6, 1 -〉 4)-linked β- d -glucopyranosyl backbone, substituted at O-3 position of (1 -〉 6)-linked β- d -glucopyranosyl by (1 -〉 3)-linked β- d -glucopyranosyl branches. ZPS consists of approximately 2930 repeating units, each contains a side chain of no more than three residues in length. Functionally, ZPS is a potent activator of B cells, macrophages and dendritic cells. Depletion of ZPS branches causes a substantial reduction in its ability not only to activate B cells in vitro but also to elicit specific IgM production in vivo. Virtually all healthy human subjects possess high-titer circulating antibodies against ZPS backbone, suggesting that ZPS epitope is shared by environmental antigens capable of eliciting adaptive humoral responses in the population.