According to the principles of green chemistry and co-efficiency, natural polysaccharides with biological activities, particularly immunoregulation and antitumor activities, have attracted increasing attention. However, the lack of information concerning the pharmacokinetics of polysaccharides is one of the major obstacles limiting their rational clinical use. In this study, triple helical β-glucan (THG), a β-1,6-branched β-1,3-glucan isolated from Lentinus edodes, was studied to clarify its cellular uptake after parenteral (e.g. intraperitoneal) administration and to determine its effect on immune cells in murine tumor models. The results obtained from size exclusion chromatography with static light scattering, differential refractometry and viscometry (SEC-LLS–RI–Vis) and atomic force microscopy (AFM) confirmed that all three THG samples displayed an extended stiff chain conformation with apparent average contour lengths of 598 nm, 510 nm and 117 nm, respectively. The ex vivo results indicated that THG directly promoted cell proliferation of peritoneal macrophages, whole spleen cells and lymphocytes, and activated peritoneal macrophages with TNF-α production. In our findings, the intraperitoneally (i.p.) administrated THG was initially ingested by peritoneal resident macrophages, which transported it to lymph nodes, thymus and even tumor. Simultaneously, THG in macrophages was biodegraded into fragments with granular shapes and small size, which were sufficiently active to be easily ingested by neutrophils. Furthermore, THG fragments could promote the infiltration of macrophages, neutrophils and DCs into tumors, and also activate these cells to enhance their cytotoxicity toward tumor cells, leading to their apoptosis. This study provides important information concerning the ingestion and processing in vivo of triple helical β-glucan from natural products, leading to a better understanding of its antitumor mechanism through activating immune cells in murine tumor models.