The purpose of this study was to study polysaccharides isolated from Polygonatum sibiricum to establish the structure-activity relationships of the active substances and to discover the optimal fraction for further development and application. Four polysaccharides fractions (PSP1, PSP2, PSP3 and PSP4) from P. sibiricum were obtained by DEAE-Sepharose Fast Flow ion-exchange chromatography. Acid hydrolysis and FT-IR spectral and NMR spectral analyses were employed for structural analysis. Our results illustrated that PSP with different chemical structure and monosaccharide composition showed different abilities to activate phagocytic activity in vitro. According to the preliminary screening results in vitro, the newly identified water-soluble polysaccharides of PSP3 were selected for further evaluation in vivo. The results demonstrated that PSP3 possessed an immunomodulatory function and could be regarded as a promising candidate as an immunomodulator.