Abstract: This study was carried out to investigate the mechanism of signal transduction in murine immunocytes induced by polysaccharide from Astragalus membranaceus. Nitric oxide (NO) concentrations of murine peritoneal macrophages were examined by a spectrophotometric assay based on the Griess reaction; cytoplasmic free Ca²⁺ in murine splenic lymphocytes was measured by a spectroflurometer assay with probe of Fura2-AM. A new ion-pair reversed phase performance liquid chromatograph was used to determine protein kinase C (PKC) activity of spleen lymphocytes in mice. The results showed Astragalus membranaceus polysaccharide increased the production of NO of macrophages and the concentration of intracellular free calcium in splenic cells and activated the PKC of spleen lymphocytes in vitro in mice. In conclusion, Astragalus membranaceus polysaccharide might perform its immunoregulation functions via signal transduction system in immunocytes by activating immunocytes to release NO so as to modulate level of intracellular free calcium and raise the activity of PKC.