Abstract: Water soluble Hohenbuehelia serotina polysaccharides(HSP)was isolated and its immunomodulatory activities in macrophage Raw264.7 cells were evaluated in vitro. Macrophage proliferation was conducted by WST-1.NO concentration was analyzed by griess reagent. Tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and prostaglandins E2(PGE₂)was detected by ELISA. And transcriptional level of iNOS,COX-2,IL-6,TNF-α and IL-1β was determined by RT-qPCR. The results indicated that HSP could significantly stimulate intracellular nitric oxide(NO)concentration in Raw264.7 cells. When the concentration of HSP was 100.00 μg/mL,the secretion of NO reached to the maximum which was 30.4335 μmol/L. Compared with blank control,HSP could significantly increase cytokines in Raw264.7 cells such as the tumor necrosis factor-α(TNF-α),prostaglandin E₂(PGE₂)and interleukin-1β(IL-1β)levels,and its secretion of high dose group(29.94 pg/mL,1.8174 ng/mL)was similar to that in the LPS positive control group. The results of RT-qPCR analysis showed that HSP also could enhance inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)expression in Raw264.7 cells. These results suggested that HSP had potential immunomodulating activities and could be applied in functional food and medicine.