Gelatin was extracted from the skin of Japanese flounder ( Palatichtys olivaceus ) and was subjected to enzymatic hydrolysis. The peptic hydrolysate resulted in a potent antioxidative peptide Gly-Gly-Phe-Asp-Met-Gly (582 Da), which bears +12.61 kcal/mol hydrophobicity. The antioxidative potential of the peptide was characterized by analyzing the protective effect of the peptide on reactive oxygen species (ROS)-mediated intracellular macromolecule damage. It was found that the peptide is a potent scavenger of intracellular ROS, thereby protecting the radical-mediated damage of membrane lipids, proteins, and DNA. Moreover, the peptide is capable of upregulating the expression of inherent antioxidative enzymes, superoxide dismutase-1, glutathione, and catalase. Collectively, it can be concluded that Japanese flounder skin, a processing byproduct of filleting, can be effectively used to produce a bioactive peptide with potent antioxidant capacity.