In this study, antioxidant peptides were prepared from defatted pumpkin seeds and purified by ultrafiltration and reverse-phase high performance liquid chromatography (RP-HPLC). Their antioxidant activity analysis and structural characterization were carried out. Moreover, their protective effects against oxidative damage in Caco-2 cells were investigated. The results indicated that 41.83% of pumpkin seed peptides had molecular masses less than 1 000 Da. The peptides had significant in vitro antioxidant activity, scavenging (91.62 ± 0.48)% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at a concentration of 40 mg/mL. The purified antioxidant peptides (at concentrations ranging from 50 to 400 μg/mL) exhibited no cytotoxic effects. The antioxidant peptides significantly increased the survival rate of H2O2-stimulated Caco-2 cells while increasing the activities of superoxide dismutase (SOD) and catalase (CAT). Additionally, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were markedly reduced, demonstrating a good protective against H2O2-indued damage in Caco-2 cells. This study provides a reference for the comprehensive development and high-value utilization of pumpkin seeds and establishes a foundation for the application and development of pumpkin seed antioxidant peptides as a novel antioxidant agent
