(1→3)-α-d-Glucan is an important component of the cell wall of most fungi. The polymer has many applications, including as a therapeutic agent in the prevention or treatment of various diseases, as well as a heavy metal sorbent and a component of new materials used in the plastics industry. The presence of (1→3)-α-d-glucan (water-insoluble, alkali-soluble polysaccharide) in the cell wall of Pleurotus djamor (pink oyster mushroom) was confirmed using specific fluorophore-labeled antibodies. Therefore, the water-insoluble fraction (WI-ASF) of P. djamor B123 fruiting bodies was isolated by alkaline extraction and used for further analyses. The structural features of the WI-ASF were determined by composition analysis, linkage analysis, Fourier transform infrared and Raman spectroscopy, 1H and 13C nuclear magnetic resonance spectroscopy, scanning electron microscopy, as well as viscosity, specific rotation, and gel permeation chromatography. These studies revealed the presence of glucose units linked by α-glycosidic bonds and scanty amounts of mannose and xylose. Furthermore, methylation analysis of WI-ASF demonstrated that the (1→3)-linked glucopyranose (Glcp) is the primary moiety (86.4%) of the polymer, while the 3,4- and 3,6-substituted hexoses are the branching residues of the glucan. The results of chemical and spectroscopic investigations indicated that the analyzed WI-ASF is a (1→3)-linked α-d-glucan type with a molecular weight of 552 kDa.