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The chemical structure of GLP-1, a novel water-soluble heteropolysaccharide purified Ganoderma leucocontextum fruiting bodies, has been characterized in our previous study. This study aimed to investigate the immunostimulatory activity of GLP-1 in vitro and in vivo by using RAW264.7 macrophages and cyclophosphamide-induced immunosuppressed mice model. Results showed that GLP-1 was able to enhance phagocytic activity and promote the production of reactive oxygen species, nitric oxide, tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 in RAW264.7 macrophages. Moreover, GLP-1 could activate mitogen-activated protein kinase, phosphatidylinositol-3-kinase/protein kinase B, and nuclear factor-kappa B signaling pathways through toll-like receptor 2 and dectin-1 receptors. Furthermore, GLP-1 increased the thymus index, serum immunoglobulin levels, and percentage of CD3+ T lymphocytes in cyclophosphamide-induced immunosuppressed mice. These results demonstrated that GLP-1 possessed significant immunostimulatory effects in vivo and in vitro and could be developed as an effective immunomodulator for application in functional foods.