Abstract: This study aims to improve the PDI and functional properties of soy protein isolate. The high-pressure homogenization combined with freeze-drying was used to prepare soy protein isolate particles. A structure-activity relationship was established to clarify the effect of pressure on the properties and structure of soy protein isolate particles after preparation. The results showed that the average particle size of soy protein isolate was greatly reduced, with the gradual increase of pressure, where the distribution curve of particle size moved to the left. The particle size of soy protein isolate was reduced by about 1 631% at 100 MPa, indicating a much wider curve distribution of particle size, compared with natural soy protein isolate. The PDI and functional properties of soy protein isolate particles were significantly improved, with the increase of pressure in the range of 60-100 MPa. The solubility of soy protein at 100 MPa, emulsifying activity, emulsification, and oil retention increased by 172.98%, 28.71%, 77.82%, and 123.76%, respectively, while the foam height also increased with time. The reason was that the unfolding of protein structure and enhanced surface hydrophobicity allowed the protein to be rapidly adsorbed at the air-water interface. Scanning electron micrograph demonstrated that there was an aggregated state in the soy protein isolate particles that had not been homogenized under high pressure, indicating that the spherical surface was recessed inward. Correspondingly, the soy protein isolate particles presented a network structure after the high-pressure combined freeze-drying process. The hydrophobic groups of soy protein isolate particles were exposed to a large amount, where the surface charge enhanced as the electrostatic repulsion increased, namely the surface hydrophobicity increased under the action of high pressure and shear force. The solubility and the improved functional properties depended mainly on the transformation of α-helix and β-turn to β-sheet and random coil structure. It was a benefit to improve the functional properties and structural characteristics of soy protein. The finding can provide a sound theoretical basis in the extensive application of the food industry.