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Armillaria luteo-virens (A. luteo-virens) is a kind of edible fungus mainly exists in Qinghai-Tibet of China, but at present only very few studies focus on the bioactivities of its polysaccharides. This study aimed to purify and characterize the structure features of a novel intracellular polysaccharide (ALP-A) derived from A. luteo-virens and explore its potential anti-tumor and immunomodulatory activities. Through systematic separation and purification, we obtained a homogeneous ALP-A with an average molecular weight of 23693Da. Structural analysis indicated that ALP-A was mainly composed of glucose and mannose with a molar ratio of 6.02:1. The repeating unit of ALP-A was →4) -α-D-Glcp-(1→ backbone with α-Glcp-(1→ and α-Manp-(6→ side chains which branched at O-2 position. The anti-tumor assays in vivo suggested that ALP-A could effectively restrain S180 solid tumor growth, protect immune organs and promote the secretion of cytokines (IL2, IL6 and TNF-α) in serum. Besides, in vitro immunomodulatory assays indicated that ALP-A could improve proliferation, phagocytic capacity and raise the level of NO and cytokines in Raw264.7 cells. These results demonstrate that ALP-A which possess potential antitumor and immunomodulatory abilities can be developed as a new functional food. [Display omitted] • Structural analysis indicated that the repeating unit of ALP-A was →4) -α-D-Glcp-(1→ backbone with α-Glcp-(1→ and α-Manp-(6→ side chains which branched at O-2 position. • The anti-tumor assays in vivo suggested that ALP-A could effectively restrain S180 solid tumor growth, protect immune organs and promote the secretion of cytokines. • In vitro immunomodulatory assays indicated that ALP-A could improve proliferation, phagocytic capacity and raise the level of NO and cytokines in Raw264.7 cells. [ABSTRACT FROM AUTHOR]