This study investigates the effects of different pressures (200, 250, 300, 350, and 400 MPa) and durations (5, 10, 15, 20, and 25 min) on the functional properties, secondary structure, and intermolecular forces of cowhide gelatin. Our results show that high hydrostatic pressure significantly affected the two, three, and four-level structures of gelatin and caused the contents of the α-helix and β-turn to decrease by 68.86% and 78.58%, respectively (p < 0.05). In particular, the gelatin at 300 MPa for 15 min had the highest gel strength, emulsification, solubility, and foaming of all the treatment conditions under study. The analysis of the surface hydrophobicity, sulfhydryl content, zeta potential, and Raman spectroscopy shows that at a pressure of 300 MPa (15 min), the hydrogen bonds and hydrophobic interactions between collagen molecules are strongly destroyed, leading to changes in the tertiary and quaternary conformation of the protein and unfolding, with the electrostatic repulsion between protein particles making the decentralized state stable. In conclusion, moderate pressure and time can significantly improve the functional and structural properties of collagen, which provides theoretical support and guidance for realizing the high-value utilization of cowhide.