Distillers dried grains with solubles (DDGS) are rich in nutrition, and they are potential protein feed raw material. However, the existence of cellulose, hemicellulose and lignin hinders animals' digestion and absorption of DDGS. Making full use of unconventional feed resources such as DDGS can alleviate the shortage of feed resources to a certain extent. This research investigated the effects of twin-screw extrusion on the macromolecular composition, physical and chemical properties, surface structure and in vitro protein digestibility (IVPD) of DDGS. The findings showed that extrusion puffing significantly increased the protein solubility, bulk density, water holding capacity, and swelling capacity, while significantly decreased hemicellulose and crude protein content, particle size and zeta potential of DDGS. The structure damage of DDGS induced by the extrusion was characterized by scanning electron microscopy (SEM), Fourier-transform infrared (FITR) spectroscopy and X-ray diffraction (XRD) analysis. Interestingly, no random coil was observed in the analysis of the secondary structure, and extrusion promoted the transformation of α-helix and β-turn to β-sheet, which led to significant increases in protein solubility and IVPD of DDGS (P < 0.05). Additionally, correlation analysis revealed that IVPD and PS had a positive relationship. Extrusion puffing was an ideal pretreatment method for DDGS modification to improve in vitro protein digestibility. © 2023 Society of Chemical Industry.