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A water-soluble polysaccharide (GLP-2) was isolated and purified from Ganoderma lucidum by the microwave-assisted freeze-thaw method. Microwave-assisted rapid thawing treatment can greatly improve the extraction yield of polysaccharides. The chemical structure of GLP-2 was analyzed with FT-IR, HPLC, GC-MS, and NMR spectrometry. It was proved that GLP-2 is mainly composed of β-1,3-glucose and β-1,6-glucose. The chain conformation of GLP-2 was analyzed by SEC-MALLS-RI, indicating that GLP-2 adopts semi-rigid chain conformation. Its molecular weight is 16.7 × 104, and its molecular size is about 61.2 nm. MTT assay indicated that GLP-2 was non-toxic to RAW 264.7 cells in vitro. The results of anti-inflammatory studies showed that GLP-2 can inhibit the production of NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage stimulated by lipopolysaccharide (LPS). The above data showed that the extraction method used in this study can obtain a high-yield polysaccharide with anti-inflammatory activity.