Gelatin is widely used in food products to improve the stability and quality of food. Different methods are currently used to alter the physicochemical properties of proteins. One of the most important of these methods is the use of heat-based methods, which in addition to the positive effects on proteins, have negative effects. For this purpose, non-thermal methods have been considered to prevent adverse effects of heat. The present study investigates the effects of using one, two and three cycles of high pressure homogenization on the functional and physicochemical properties of gelatin at concentrations of 3%, 6% and 9%. In this regard, after homogenization of gelatin at pressure 1000 bar of gel stiffness, color, emulsifying capacity and emulsion stability, foaming and foaming stability, free sulfhydryl content, infrared spectrum, electrophoresis pattern, visible-ultraviolet spectrophotometry, secondary structure and Microscopic microstructures were studied. The results of this study showed that the effect of high pressure homogenization on gel stiffness depends on its concentration, at 3% concentration this process has no significant effect on gel stiffness but at 6% and 9% concentrations it causes tissue weakening. Change in the three factors of yellowness, brightness and redness of gelatin also depend on the gelatin concentration and the number of homogenization cycles. The use of high pressure homogenization also significantly increased the emulsification capacity, emulsion stability and sulfhydryl release, but with increasing number of homogenization cycles the emulsifying capacity, emulsion stability and free sulfhydryl decreased (p 0.05). This process can change the amount of foaming, by performing high pressure homogenization, the gelatin content decreases, However, with increasing number of homogenization cycles, there was a significant increase in the amount of foaming (p 0.05), But this process had little impact on the stability of the foaming. Infrared spectroscopy and circular dichroism spectrometer show gelatin denaturation and protein ?-helical opening due to high pressure homogenization. The results of UV-visible spectrophotometry also confirm these results. Although the results of the second and third structures show changes in these structures after homogenization, the examination of the processed gelatin electrophoresis pattern showed that high pressure homogenization could not effect the gelatin chain length. The microstructure study of this protein also revealed the conversion of gelatin sheet structure to polyhedral structures as a result of this process, Also the cavities in the structure were smaller but their wall thickness varied depending on the gelatin concentration and the number of homogenization cycles. Overall, the results showed that the use of high pressure homogenization as a non-thermal method significantly improved the functional properties of gelatin due to the change in the second and third gelatin structures. Keywords : High pressure homogenization, Functional propertied, Protein structure, gelatin