In this study, we investigated the protective effects of a Neutrase enzymatic hydrolysate derived from Korea pen shell Atrina pectinata (APN) against hydrogen peroxide (H2O2)-induced oxidative damage in hepatocytes. First, we confirmed that APN has antioxidant activities by scavenging 2,2-azino-bis (3-ethylbenzthiazoline)-6-sulfonic acid radical (ABTS+) and H2O2 and increasing oxygen radical absorbance capacity (ORAC) value. Also, the treatment of APN increased the cell viability by reducing the intracellular reactive oxygen species (ROS) production in H2O2-stimulated hepatocytes. In addition, APN decreased the sub-G1 DNA contents and the apoptotic body formation increased by H2O2 stimulation. Moreover, APN modulated the protein expression of apoptosis related molecules (Bcl-2, Bax and p53) by suppressing the activation of nuclear factor NFkB and ERK/p38 signaling in H2O2-stimulated hepatocytes. Furthermore, APN led to the activation of Nrf2/HO-1signaling known as antioxidant systems. These results suggest APN protects hepatocytes against oxidative damages caused by H2O2 stimulation.