Abstract: To elucidate the mechanism underlying the inhibition of beef protein oxidation by lotus seedpod proanthocyanidin (LSPC), epicatechin gallate (ECG; a key bioactive component of LSPC) at a final concentration of 10 to 40 μmol/L, was investigated using a combination of nanoparticle size analysis, two-dimensional infrared correlation spectroscopy (2D-IR COS), and fluorescence spectroscopy. This study focused on how ECG affected the oxidative aggregation of beef myosin as well as the interaction mode, dominant binding forces, action sites, involved functional groups, and fluorescence quenching mechanism between ECG and myosin. The results indicated that, relative to native myosin, ECG treatment at final concentrations of 10~30 μmol/L resulted in a narrowed 250 kDa band in the sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles of the ECG-Myosin complex, an increased absolute value of zeta potential, and reduced turbidity. These observations suggested that ECG inhibited oxidative myosin cross-linking with non-covalent interactions between ECG (acting as a hydrogen donor) and myosin. Fluorescence spectroscopy and chemical probing analyses revealed that ECG binding to myosin was an entropy-driven, thermodynamically favorable endothermic process, with hydrogen bonds and hydrophobic interactions serving as the dominant forces. This interaction induced the static fluorescence quenching of myosin. Furthermore, 2D-IR COS demonstrated that the hydroxyl (-OH) groups on the D-ring of ECG, as hydrogen donors, preferentially formed hydrogen bonds and hydrophobic interactions with carbonyl (C=O), amide (N-H), and alkyl (C-H) groups within the α-helical domains of myosin. These interactions inhibited protein oxidation and intermolecular cross-linking aggregation. This study provides a theoretical basis and experimental support for LSPC development as a natural plant-derived antioxidant for meat and meat products industry-related applications.