The nonstarch polysaccharides (NSPs) in the total dietary fiber (TDF) from the sclerotia of Pleurotus tuber-regium (tiger milk mushroom) were fractionated by the sequential use of chemical solvents. About half of the TDF was solubilized and two major alkali-soluble fractions (1 and 4 N sodium hydroxide) that contained 126 and 293 g/kg TDF were obtained. Sugar analysis and infrared spectroscopy indicated that the NSPs in these alkali-soluble fractions were mainly beta-glucans and chitin. These alkali-soluble NSPs were further purified by anion-exchange chromatography followed by gel permeation chromatographic separation. Methylation analysis revealed that these purified glucans were highly branched and contained a mixture of sugar linkages of beta-1,3, beta-1,6, and beta-1,4. The potential use of these sclerotial beta-glucans as nutraceuticals was discussed.