Whole wheat flour (WWF) prepared by the direct crushing method preserves all the components of the whole wheat grain. WWF with different particle sizes (180, 150, 125, 106, and 96 μm) was obtained by combining stone milling and particle size sieving technology. The effects of particle size on the proximate composition, farinograph, pasting, thermal, and functional properties, starch microstructure, and Fourier-transform infrared (FTIR) spectroscopy of stone-milled WWF were investigated. The smaller the particle size of WWF, the higher the damaged starch content. The water absorption, degree of softening, pasting temperature, solubility, and syneresis of WWF increased steadily as the particle size decreased, whereas the peak viscosity, final viscosity, swelling power, water holding capacity, and enthalpy of gelatinization decreased. The scanning electron microscope micrographs revealed that the larger the particle size of WWF, the denser the distribution of starch granules. The β-sheet and β-turn contents of WWF with particle size 180 μm were the highest, reaching up to 33.85% and 39.79%, respectively. The particle size exerted influence on the quality characteristics of stone-milled WWF, and the overall properties of WWF were better at medium particle size. © 2023 Society of Chemical Industry.