Chitin-glucan complex (CGC) is a novel insoluble dietary fiber with multiple physiological activities. In this work, CGC was extracted from the fruiting body of Coprinus comatus and its physicochemical properties and prebiotic effects were investigated. The results indicated that CGC consisted of glucosamine and glucose in a molar ratio of 67: 33 with degree of acetylation of 61.91% and crystallinity index of 25.40%. The maximum degradation temperature was determined to be 307.52 °C, and a woven fibrous structure was observed by scanning electron microscopy. CGC exhibited higher oil-holding capacity, water-holding capacity and nitrite ion adsorption capacity than commercial chitin, and showed potential prebiotic effects. Compared with control and commercial chitin, CGC significantly (P < 0.05) increased the concentration of propionic and butyric acids. These results suggested that CGC from C. comatus was promising to be an alternative source of CGC products and used as a bioactive ingredient in functional foods.