In this study, crude extract of the seagrass Zostera japonica, and its solvent-partitioned fractions were evaluated for their antioxidant activity. The crude extract was successively fractionated into n-hexane, 85% aqueous methanol (85% aq.MeOH), n-butanol (n-BuOH), and water fractions by liquid-liquid partition. These include DPPH radical scavenging, hydroxyl radical scavenging in HT-1080 cells, peroxynitrite scavenging, and protective effect on DNA damage caused by hydroxyl radicals generated. In all assays, except for DPPH radical, 85% aq.MeOH and n-BuOH fraction showed the strong antioxidant activity. These results suggest that Z. japonica may be used as a potential source of natural antioxidants for the development of cosmetic product or functional food in the future.