Numerous polysaccharides isolated from plants have been used to augment traditional drugs in the treatment of cancer. In order to explore the influence to hepatocellular carcinoma, a novel cold water-soluble polysaccharide was separated from Rhodiola rosea L. root (RLP) and then its structure and anti-cancer activities were tested. The chemical compositions and high performance gel permeation chromatography (HPGPC) results indicated that RLP was an acid heteropolysaccharide with the molecular weight of about 1.15×106 Da. Furthermore, ion chromatography (IC), Fourier transform infrared (FT-IR) and nuclear magnetic resoance (NMR) further indicated that RLP was main composed of →2,4)-α-Rha(1→, →5)-α-L-Araf-(1→, α-D-Glu, →6)-β-D-Galp-(1→, β-D-Man and →4)-α-GalpA-(1→. In vivo antitumor activities of RLP were carried out by using H22 tumor-bearing mice model. The results shown that RLP (100 and 300 mg/kg) could inhibit tumor growth of H22 cells from 23.59% to 45.52% and protect thymuses and spleen without damage. In addition, according to cell cycle, AV-FITC/PI and JC-1, RLP could induce dose-dependent apoptosis of H22 cells via S phase arrested which was through a mitochondrial related pathway. Our data indicated that RLP has a broader application prospect in anti-tumor preparations.