A novel Pleurotus nebrodensis polysaccharide(PN-S) was purified andcharacterized,and its immune-stimulating activity was evaluated in RAW264.7 macrophages.PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner,as determined by the MTT assay.After exposure to PN-S,the phagocytosis of the macrophages was significantly improved,with remarkable changes in morphology being observed.Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases.PN-S treatment enhanced the productions of interleukin-6(IL-6),nitric oxide(NO),interferon gamma(INF-γ),and tumor necrosis factor-α(TNF-α) in the macrophages,with up-regulation of m RNA expressions of interleukin-6(IL-6),inducible nitric oxide synthase(i NOS),interferon gamma(INF-γ) and tumor necrosis factor-α(TNF-α) being observed in a dose-dependent manner,as measured by q RT-PCR.In conclusion,these results suggest that the purified PN-S can improve immunity by activating macrophages. A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was as in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose- dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2 / M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF- γ), and tumor necrosis factor-α of m RNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma (INF-gamma) and tumor necrosis factor- as measured by q RT-PCR.In conclusion, these results suggest that the purified PN-S can improve immunity by activating macr ophages.