In this study, cathepsin D was oxidized in vitro with different concentrations of H2O2, and the activity, structure, and extent of myofibrillar protein degradation by oxidized cathepsin D were evaluated. The sulfhydryl content of cathepsin D decreased to 9.20% after oxidation, while the carbonyl content increased to 100.06%. The β-sheet in the secondary structure altered due to oxidation as well. The changes in the intrinsic fluorescence and UV absorption spectra indicated that oxidation could cause swelling and aggregation of cathepsin D molecules. The structure of cathepsin D could change its activity, and the activity was highest under 1 mM H2O2. Cathepsin D could degrade myofibrillar proteins in different treatment groups, and the degree of degradation is various. Therefore, this study could provide a scientific basis for the mechanism of interaction among hydroxyl radical oxidation, cathepsin D, and MP degradation.