Abstract: This study aimed to investigate the impact of particle size changes on the physicochemical properties and in vitro digestion characteristics of wheat granular flour starch system. By controlling the number of grinding times and without sieving treatment, the particle size of the granular flour, initially 224 µm, was successively reduced to 115, 91, 80, 68 and 62 µm. The study systematically explored the changes in starch physicochemical properties and in vitro digestion characteristics induced by particle size changes and preliminarily explored the underlying mechanisms based on the multi-scale structure of starch. The results showed that as the particle size of wheat granular flour gradually decreased, the damaged starch content significantly increased from 0.71% to 13.19% (P<0.05). In terms of pasting properties, the peak viscosity first significantly rose to 2.50 kcP (P<0.05) and then significantly dropped to 1.96 kcP (P<0.05). The retrogradation value significantly decreased to 0.96 kcP (P<0.05). Comprehensive analysis of the changes in pasting and thermodynamic properties of wheat granular flour induced by particle size changes indicated that when the particle size of wheat granular flour was in the range of 80~115 µm, the degree of expansion was the largest. In the in vitro simulated small intestine digestion test, when the particle size of wheat granular flour was reduced to 80 µm and below, the digestion rate constant k value (0.032) of starch molecules and the glucose release amount (26.65 g/100 g starch) after 2 hours of digestion significantly increased (P<0.05). However, these values were still significantly lower than those of the digestion rate constant k value (0.049) and glucose release amount (59.70 g/100 g starch) of commercial refined wheat flour. Further research on the multi-scale structure of starch using scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy found that when the particle size of wheat granular flour was reduced to 80 µm and below, the release amount of free starch granules significantly increased, and the crystalline regions, short-range order and double helical structure of starch molecules were obviously damaged. Therefore, controlling the particle size of wheat granular flour in the range of 91~115 µm is expected to be applied to the production of slow-digesting foods, which can help control blood glucose in the human body and reduce the incidence of chronic diseases such as obesity and diabetes.