A novel polysaccharide AAMP-A70 (5.6 kDa) has been purified from the fruiting bodies of Amillariella mellea. Compositional analysis and 1H and 13C NMR spectra indicate that AAMP-A70 is a branched β-glucan with a main chain that consists of β-d-(1→6) linked Glcρ residues substituted at O-3 by β-Glcρ or α-d-(1→6)-linked Galρ side chains. AAMP-A70 increases macrophage phagocytosis and secretion of NO, ROS, TNF-α, IL-6 and IL-1β. Mechanistically, AAMP-A70 promotes degradation of IκB-α and nuclear translocation of the NF-κB p65 subunit, and enhances phosphorylation of MAPKs. In particular, the function blocking antibody to TLR2 substantially suppresses TNF-α and IL-6 production. Our data demonstrate that AAMP-A70 activates macrophages via NF-κB/MAPK signaling pathways and the TLR2 receptor. Overall, AAMP-A70 may serve as a good food supplement to enhance immunity.