Abstract: In this study, the crude fucoidan extracted from Kjellmaniella crassifolia was taken as raw material, and then purified by DEAE-Sepharose Fast Flow weak anion exchange column. The contents of total polysaccharide and sulfate ( SO₄^2-) of fucoidan in each fraction were determined by phenol-sulfuric acid colorimetry method and barium chloride-gelatin method, respectively.The antitumor activities for all in the crude and separated fractions with 50, 200, 800 μg/m L concentrations against Hep G2 cells, A549 cells and HT-29 cells were determined by MTT assay. The results showed that, crude fucoidan from Kjellmaniella crassifolia could be well separated and purified by DEAE-Sepharose Fast Flow weak anion exchange column, and five different fractions ( F-0, F-1, F-2, F-3, F-4) were obtained. Total polysaccharide and SO₄^2- content in F-4 were the highest, which were 64.25% and 26.32%, respectively. The results of MTT assay indicated that the crude fucoidan and the fractions had significant inhibitory activities on Hep G2, A549 and HT-29 cells.The lowest cell viability of 67.69%, 56.03% and27.56% were obtained for Hep G2, A549 and HT-29 cells respectively.Based on those findings, it suggested that fucoidan from Kjellmaniella crassifolia had great potential to be a kind of effective natural antineoplastic drugs.