A sulfated fucoidan named as SF0, was isolated from Sargassum pallidum with hot water extraction and further purified by a strong anion exchange column and a gel filtration column. SF0 was further fractionated into three fractions SF1, SF2 and SF3 by stepwise partial acid hydrolysis. The content of sulfated group and molecular weight were decreased from SF0 to SF3. The structural features and anti-influenza A (H1N1) virus activities of all these fractions were determined. Results showed that SF0 was a homogeneous polysaccharide with a large molecular weight of 628.1 ku. It was mainly composed of mannose, glucuronic acid, glucose, galactose, xylose and fucose, in the molar ratio of 10.8∶8.3∶3.0∶21.1∶21.2∶35.6, SF0 has a backbone composed of α-D-1,2-Manp and β-D-1,4-GlcAp disaccharide repeat unit. The anti-influenza A (H1N1) virus assay showed that all these fractions inhibited H1N1 virus replication in MDCK cells. Moreover, the fraction SF3 with the low molecular weight had better activity than original fraction. It suggested that the anti-H1N1 virus activity of fucoidan was related to its fine structure and molecular weight.