Abstract: The present study was aimed to investigate the structural characteristics and immunomodulatory activity of Suillus granulatus polysaccharides. Two polysaccharide fractions(SGP3-1 and SGP3-2) were purified from the fruiting bodies of Suillus granulatus by water extraction and alcohol precipitation, deproteinization, and column chromatography separation. The physicochemical properties of SGP3-1 and SGP3-2 were characterized by high-performance gel permeation chromatography(HPGPC), high-performance liquid chromatography(HPLC), Fourier transform infrared spectroscopy(FT-IR), nuclear magnetic resonance spectroscopy(NMR), X-ray diffraction(XRD), etc. The results showed that the weight-average molecular weight of SGP3-1 and SGP3-2 were 170.48 and 14.52 kDa, respectively. Both SGP3-1 and SGP3-2 were low-crystallinity amorphous polymers, and had no triple-helix conformation. Additionally, SGP3-1 characterized by α-type glycosidic linkage was mainly composed glucose(55.47%), mannose(20.63%) and galactose(14.04%), while SGP3-2 characterized by β-type glycosidic linkage was mainly composed of glucose(82.43%). The results from RAW264.7 cells in vitro suggested that SGP3-1 and SGP3-2 showed no cytotoxicity in the concentration range from 5 to 40 μg/mL. Moreover, SGP3-1 and SGP3-2 could significantly enhance the phagocytic activity and promote the production of nitric oxide(NO), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) through activating mitogen-activated protein kinases(MAPKs), phosphatidylinositol-3-kinase(PI3K)/Akt, and nuclear factor-κB(NF-κB) signaling pathways. Furthermore, SGP3-2 exhibited better immunomodulatory activity than SGP3-1. It showed that SGP3-1 and SGP3-2 could be used to develop functional foods, as dietary supplements for people with low immunity, and provide a theoretical basis and research basis for the further development of Suillus granulatus.