Abstract: The relationship between α-glucosidase inhibitory activity and the composition bond type of binding protein of high temperature water-extracted polysaccharides ( HIOP) that extracted from Inonotus obliquus was studied.HIOP was enzymatically digested with four different proteases, and the effect of protease hydrolysis on its molecular weight composition andα-glucosidase inhibitory activity was determined. The inhibitory rate of α-glucosidase by original HIOP was 83.72% at10 μg/m L, while the lowest α-glucosidase inhibition rate of HIOP that hydrolyzed by neutral protease, alkaline protease, pepsin, and trypsin were 53%, 65%, 6.5% and 7.1%, respectively. The binding protein in HIOP had a role in theα-glucosidase inhibitory activity of HIOP.In addition, in consideration of the effects of these four kinds of enzymes, it could be speculated that the active sites of the binding proteins in HIOP contain aromatic amino acids, acidic amino acids, lysine or arginine.If we destroy such peptide bonds, the α-glucosidase inhibitory activity of HIOP could be significantly reduced.But, the four proteases did not change the molecular weight of HIOP, indicating that the four proteases only affected the binding site of HIOP to α-glucosidase activity center.