Polysaccharides fromArmillaria luteo-virens (ALP) were investigated for structural characterization and immunomodulatory activities. Three fractions (ALP-1, ALP-2, and ALP-3) were obtained with the yield of 2.4, 3.7, and 3.0 wt %, respectively. ALP-1 was proposed as a β-(1 → 3)(1 → 6)-glucan with a triple-helix conformation; ALP-2 and ALP-3 were both identified as α-(1 → 4)(1 → 6)-glucan differing in their M w and branching degree with a spherical conformation. The in vitro digestibility experiment and in vivo experiments using cyclophosphamide (CY)-treated mice demonstrated that intraperitoneal injection of α-glucan (1 mg•kg −1 •day −1 ) and intragastric gavage of β-glucan (10 mg•kg −1 •day −1 ) both effectively restored the decrease in body weight, immune organ indexes, immune cell activities, serum immune marker levels, colonic short-chain fatty acids (SCFA) levels, and Bacteroidetes/Firmicutes ratio in immunosuppression mice. This study provides novel insights into the immunomodulatory activity of αand β-glucans under different administration routes, thereby promoting their application in both food and pharmaceutical areas.