The microstructure of collagen from pig skin was studied during gelatinization. The results showed that a relatively high degree of gelatinization of collagen was achieved after 8-h acid treatment and the yield of gelatin reached 83.98% after hot water extraction, while the yield of gelatin dropped significantly with increasing duration of acid treatment. The results from differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), and circular dichroism (CD) revealed that the triple-helical structure of collagen became progressively loose and valence bonds stabilizing the triple-helix were gradually destroyed during the process of acid treatment. The triple-helical structure was excessively destroyed after 8-h acid treatment, which could be the main reason for the decrease in gelatin yield. Moreover, the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that collagen peptide chains were degraded during acid treatment and the gelatin subunit content was directly influenced by the amount of subunits that remained after gelatinization. Therefore, during the gelatinization of collagen, a balance between subunit degradation and the loosening of the triple-helical structure is required, in order to obtain high yield of high-quality gelatin.