Eisenia bicyclis (Kjellman) Setchell is a common brown alga that inhabits the middle Pacific coast around Korea and Japan. In this study, the ethanol extract and its serial solvent fractions were prepared from fresh E. bicyclis, and their hepatoprotective effects were investigated against hepatotoxicity in tert-butyl hyperoxide(t-BHP)-injured HepG2 cells. When these samples were used at a dose of 10-40 mu g/mL, they significantly protected the t-BHP-induced cell death in HepG2 cells. Among fractions, ethyl acetate fraction (EF) and n-butanol extract (BF) exhibited potent hepatoprotective activities (62.60% for EF and 64.86% for BF) in t-BHP-injured HepG2 cells at a concentration of 10 mu g/mL. To find the potential factors for this activity, the samples were characterized on total phenolics, chlorophylls, carotenoids, and radical scavenging activity. Among them, EF showed the highest content of total phenolics and the strongest antioxidant activity both in on- and offline assays. Five phlorotannin compounds, oligomers of phloroglucinol, were isolated chromatographically from this fraction and structurally identified by (1)H-NMR and liquid chromatography-electrospray ionization-mass spectrometry analyses as eckol(1), 6,6'-bieckol(2), 8,8'-bieckol(3), dieckol(4), and phlorofucofuroeckol A(5). Compound 5 among five purified compounds showed the strongest protective activity (45.54%) at a concentration of 10 mu M. At the high dose (40 mu M), the protective activities of three compounds (compound 2, 4, and 5) were higher than that of quercetin treated with 10 mu M concentration. Therefore, we can speculate that they can be developed as potential candidates for natural hepatoprotective agents.