Previously we showed that the underlying mechanism of oxidative stress-induced apoptosis is nuclear loss of DNA repair protein Ku70 and Ku80, which are involved in the DNA repair process of double-strand breaks. Lycopene acts as an antioxidant and a singlet oxygen quencher. In the present study, we aim to investigate whether lycopene protects oxidative stress-induced cell death of pancreatic acinar AR42J cells by preventing the loss of Ku70 in the nucleus. The cells received oxidative stress caused by glucose oxidase acting on β-D-glucose (glucose/glucose oxidase) and were cultured in the absence or presence of various concentrations of lycopene. Viable cell numbers, the levels of H2O2 in the medium, level of Ku70 protein, and Ku-DNA-binding activity were determined. As a result, glucose/glucose oxidase induced the decrease in cell viability, increase in H2O2 production, decrease in Ku70 levels in whole-cell extracts and nuclear extracts, and decrease in Ku-DNA-binding activity of AR42J cells. Lycopene inhibited glucose/glucose oxidase-induced cell death by preventing nuclear loss of Ku70 and a decrease in Ku-DNA-binding activity of AR42J cells. In conclusion, lycopene may be beneficial for the treatment of oxidative stress-induced cell death by preventing loss of DNA repair protein Ku70.