Crude polysaccharide obtained from Saccharina japonica using acid hydrolysis and precipitation was separated into sulfated fuco-oligosaccharide (HDF1) and heteropolysaccharide (HDF2). To further explore the bioactive fraction, HDF2 was successfully separated using membrane filtration into HDF2A and HDF2B, which differed in chemical composition and molecular weight. The bioactivity of all the fractions was tested in vitro, including immunomodulatory activity in RAW 264.7 cells and the protective activity in aristolochic acid (AA)-induced NRK-52E cell injury. HDF1 and HDF2B (low-molecular weight sulfated fucans/fuco-oligosaccharides) did not increase the nitric oxide production in RAW 264.7 cells, whereas HDF2 and HDF2A exhibited potential immunomodulatory activity. All the tested compounds showed different degrees of protective activity in AA-induced injury; HDF2A exhibited superior protective activity. Through chemical analysis, HPLC analysis, and IR spectroscopy and MS, it was determined that HDF2A was a galactose-enriched heteropolysaccharide- glucofucogalactan with a distinctive 2:1 ratio of galactose to fucose, In addition, HDF2A also contained a high amount of glucose and minor amounts of mannose, rhamnose, and xylose, with a low content of sulfate. Thus, HDF2A, a complex heterogeneous polysaccharide mixture with a unique monosaccharide composition, could be studied for further structural characterization and pharmaceutical applications. (C) 2018 Elsevier B.V. All rights reserved.