A new polysaccharide (IHP-1aa) was isolated from the fruiting body of Inonotus hispidus by hot water extraction, ethanol precipitation and column chromatography. The molecular weight of IHP-1aa was 26.9 kDa. Structural analysis showed that IHP-1aa consisted of glucose (Glc), galactose (Gal), fucose (Fuc), mannose (Man) and contained a certain amount of 3-O-methylgalactose (3-O-Me-Gal). The structure was mainly composed of →6)-α/β-D-Glcp-(1→, →6)-α-D-Galp-(1→, →6)-(3-O-Me)-α-D-Galp-(1→, →6)-α-D-Manp-(1 → and →2, 6)-α-D-Galp-(1 → as the main chain. Branched at O-2 with single β-L-Fucp-(1 → 6)-α-D-Galp-(1 → 6)-α-D-Glcp-(1 → as major the side chain. The results of SEM, XRD and AFM combined with Congo red indicated that IHP-1aa may be amorphous granular chain conformation. In addition, IHP-1aa stimulated macrophage function and improved phagocytic ability of RAW264.7, as well as promoted the secretion of NO, TNF-α and IL-6. IHP-1aa, a 3-O-methylgalactose-containing heteropolysaccharide, was isolated for the first time from the I. hispidus, which may be used as a potential immunomodulator in functional foods.