While the methylotrophic yeast Pichia pastoris enables the industrial‐scale biosynthesis of many recombinant products, large amount of nutrient‐rich biomass emerged along this process. Polysaccharides, especially glucans that are abundant in the cell wall of P. pastoris, are yet to be better utilized owing to their various biological activities. However, the isolation and purification of cell wall glucan from P. pastoris has not been reported. In this study, we established an environment‐friendly approach, including induced autolysis, hot‐water treatment, ultrasonication, isopropanol extraction, and protease treatment, to isolate and purify glucan from the cell wall of P. pastoris. We achieved a purity of 85.3% and a yield of 11.7% for the purified glucan. Proteins, nucleic acids, lipids, and ash were efficiently removed during the purification. The activities of the purified glucan were investigated in mice fed with a high‐fat diet. The purified glucan decreased the level of total cholesterol and triglycerides by 30.3 and 29.7%, respectively. This result suggested that the cell wall glucan of P. pastoris could be developed to a therapeutic agent for dyslipidemia. Our study proposed an environment‐friendly and effective method to isolate and purify the glucan from P. pastoris, providing solid foundation for the high‐value utilization of this yeast.