The aim of this study was to investigate the possible mechanism for increasing the antioxidant activity on peptide Glutamine-Tryptophan-Phenylalanine-Histidine (QWFH) from pine nut (Pinus koraiensis) protein by a pulsed electric field (PEF). The antioxidant capacity of PEF-treated QWFH increased significantly (p < 0.05) through 1,1-diphenyl-2-pycryl-hydrazyl radical scavenging and oxygen radical absorbance capacity assays. A series of mechanism exploration methods, including reversed-phase high-performance liquid chromatography, ultraviolet absorption spectroscopy, intrinsic fluorescence spectra, circular dichroism spectroscopy, and 1D and 2D nuclear magnetic resonance spectroscopies, were applied. QWFH chain was not cleaved by the PEF treatment, while more aromatic amino acids (Trp and Phe) were exposed to the polar solvent. In addition, the content of random coil of QWFH in solution was increased and its active hydrogen was changed after the PEF treatment. Moreover, the long-range connectivity between OH (14.234 ppm) on 4-H His, N_α H (7.295 ppm) on 3-H Phe, and N_α H₂ (6.801 ppm) on 1-H Gln disappeared due to the PEF. PRACTICAL APPLICATIONS: Antioxidants have been extensively explored as a potential drug to decrease the risk of certain chronic diseases. Food-derived bioactive compounds are safer than synthetic antioxidants for human health and well-being. And the PEF technology is one of the promising processes for improving the biological activity of food components. Currently, the activity of the antioxidant peptide QWFH increased after a PEF treatment. The basic structure of QWFH did not change, but the unfolding of the secondary structure on the peptide chain and the displacement of the active hydrogen increased the antioxidant activity of the peptide. Thus, the range of application of a PEF has been expanded and it also benefited the development of more functional factors in the functional food industry.