The aim of this study was to investigate the antiproliferative effects of fucoidan from three regional hijiki (Hizikia fusiforme) samples (Zhejiang-China, Jeju-Korea [JH], and Wando-Korea) in East Asia. Hijiki was processed using 1% citric acid to decrease heavy metal content. The JH sample was separated using diethylaminoethyl-cellulose-ion exchange chromatography to obtain four active fractions (JHCF1-JHCF4) and their monosaccharide composition was detected using high-performance liquid chromatography. The structure of the crude polysaccharides and four fucoidan fractions was analyzed using Fourier-transform infrared spectroscopy. JHCF4 showed the highest fucose and sulfate content and decreased Hep3B cell growth in 48 h with a half-maximal inhibitory concentration of 33.53 ± 2.50 μg/ml, which represented the strongest anticancer activity. Further, nuclear staining with Hoechst 33342 and acridine orange-ethidium bromide staining demonstrated that the anticancer activity of JHCF4 was mediated by apoptosis. Moreover, JHCF4 down-regulated B-cell lymphoma extra-large, while up-regulating Bcl-2-associated X protein, caspase-3, and apoptotic bodies to different degrees in Hep3B cells. JHCF4 induced apoptosis via the generation of reactive oxygen species along with the concurrent loss of mitochondrial membrane potential, indicating the potential role of the mitochondria-mediated pathway. Therefore, these results indicate that JHCF4 exhibited antiproliferative effects on the investigated cancer cell lines.