HSP-III, a novel homogeneous polysaccharide with 513.89 kDa molecular weight, was fractionated from submerged cultures of Hirsutella sinensis by Sevag and chromatography on Sephadex G-100 column. The total sugar content of HSP-III was amounted to 89.87%. Based on the results of high performance gel permeation chromatogram (HPGPC), FT-IR, NMR spectroscopy, GC, periodate oxidation-smith degradation and methylation analysis, it showed that HSP-III was mainly composed of mannose and galactose, and a small amount of rhamnose, arabinose, xylose, and glucose. The molar ratio of Rha:Ara:Xyl:Man:Glu:Gal was 1.00:2.44:13.11:74.13:13.80:54.39. The main chain of HSP-III was majorly composed of (1→3) glucose. The tumor inhibition ratio on H22 cell was 79.04% at 100 μg/mL of HSP-III.