Abstract: Currently, food protein fibrils had garnered significant attention due to their enhanced functional properties compared to natural proteins, and its formation is influenced by endogenous and exogenous factors. To broaden its application in the food industry, this paper explored the impact of high-speed shearing homogenization pretreatment on the formation and structural characteristics of soy protein isolate fibrils, which were prepared at different shear rates (0, 5000, 10000, 15000, and 20000 r/min) under the conditions of soy protein isolate fibrils (SPIF). The determination of thioflavin T fluorescence, surface hydrophobicity, particle size zeta potential, rheological characteristics, transmission electron microscopy and fourier transform infrared spectroscopy was employed to investigate the effect of shear pretreatment on the physicochemical properties, rheological characteristics and structure of SPIF. The results indicated that after shear pretreatment, compared to the unsheared pretreated samples, the thioflavin T fluorescence intensity and β-sheet content of SPIF were significantly enhanced (P<0.05) following shear pretreatment, the highest fluorescence intensity and β-sheet content were observed at a shear rate of 15000 r/min. The surface hydrophobicity and intrinsic fluorescence intensity of SPIF decreased. The particle size and turbidity decreased, while the zeta potential initially increased and then decreased. A significant increase (P<0.05) in both viscosity and modulus was observed in the rheological measurements, with peak values recorded at a shear rate of 15000 r/min. Transmission electron microscopy observed that the SPIFs behaved as semi-flexible fiber aggregates. The above results indicate that high-speed shear pretreatment has a promotional effect on the formation of SPIF and that shear pretreatment is an effective physical treatment. This work may contribute to a more profound comprehension of the effect of shear preconditioning on the formation and structural characterization of SPIF.