Abstract: The crude polysaccharide of Phellinus igniarius mycelia was prepared by the method of water extraction and ethanol precipitation. Then it was preliminarily fractionated by ammonium sulfate solution with the final concentration of 30%, 45% and 60%, respectively. The antioxidant and anti-tumor activities of the polysaccharides fractions were tested and compared with each other. The fraction which showed the strongest activity was further purified by ion exchange resin column chromatography. And the homogeneity and average molecular weight (M_W) of purified polysaccharides were evaluated and determined by size-exclusion chromatography combined with multi-angel laser light scattering and refractive indexdetector (SEC-MALLS-RI). The results showed that 45% ammonium sulfate solution supernatant fraction (IPS45) had the strongest anti-tumor activity and better scavenging superoxide anion activity in fractions fractionated by different concentrations of ammonium sulfate. Four fractions were obtained from IPS45 by DEAE-52 cellulose ion exchange column purification. SEC-MALLS-RI analysis results showed that the ddH₂O elution fraction (IPS45-W) was a homogeneous polysaccharide with a M_W of 79.1 kDa. GC analysis indicated that IPS45-W was composed of mannose:glucose:galactose:unknown monosaccharide in a molar ratio of 1.57:8.38:1.09:1.00. The IPS45-W could inhibit the growth of HepG2 cells. Its inhibition rate was 56.74% at the concentration of 50 μg/mL. This inhibition rate of IPS45-W was higher than that of the homopolysaccharide (11%~35%) against HepG2 cells at same concentration, which from the Phellinus igniarius myceli polysaccharide fractionated by ethanol precipitation in previous studies. The results would provide the basis for the separation and application of different components of Phellinus igniariusmycelia polysaccharides.