The antioxidant peptide Met-Met-Cys-Thr-Asp (MMCTD) obtained in the previous study was analyzed in this work, and 2,2-diphenyl-1-picrylhydrazyl (DPPH?) radical-scavenging capacity served as an indicator of antioxidant activity. Mid-infrared spectroscopy (MIR), circular dichroism (CD) spectroscopy, zeta-potential, and nuclear magnetic resonance (NMR) were used for an in-depth analysis of the effects of a pulsed electric field (PEF) on the antioxidant activities of peptide MMCTD. The results showed that when the electric field intensity was 5 kV/cm and pulse frequency was 2400 Hz, the DPPH radical-scavenging capacity reached a peak value (94.27%±0.03%, mean±SD) and the zeta-potential of peptide MMCTD being treated was reduced to 10.85±0.98 mV. The MIR and NMR results revealed that after the PEF treatment, the functional groups of peptide MMCTD were not changed, but the chemical environment of the hydrogen ion (proton) was changed. The CD analysis indicated that the proportion of the β-sheet in the secondary structure decreased from 13.7% to 0%, and the relative amounts of the β-turn and random coil increased significantly. These changes not only preliminarily reveal that PEF can enhance the antioxidant activity of peptide MMCTD by changing the secondary structure and surface charge distribution but also lay the foundation for identification of the mechanism underlying the enhancement of antioxidant activity by PEF.