Protein derived from the sand eel, Hypoptychus dybowskii, was hydrolyzed using different proteases (alcalase, neutrase, alpha-chymotrypsin, papain, pepsin, and trypsin) to produce an antioxidant peptide. Antioxidant activity of hydrolysates was evaluated using DPPH radical scavenging activity. The papain hydrolysate exhibited the highest antioxidative activity compared to other hydrolysates. The free radical scavenging activity of papain hydrolysate was 77.4% at 1.0 mg/ml. The peptide demonstrating the strongest antioxidative activity was isolated from the hydrolysate using consecutive chromatography. The amino acid sequences of purified peptide was identified as Ile-Val-Gly-Gly-Phe-Pro-His-Tyr-Leu (1189 Da), and the EC(50) value of antioxidant peptide was 22.75 mu M. The purified peptide exhibited an inhibitory effect against DNA oxidation induced by hydroxyl radical. The results of this study suggest that sand eel protein hydrolysate is a good source of natural antioxidants. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.