Abstract: In this study, a single-screw extruder was used to extrude distiller's grains (DGs) and the effect of extrusion material moisture (21%, 24%, 27%, 30%, 33%) on prolamins from DGs (PDGs) was studied by measuring the physicochemical (solubility, hydrophobicity, sulfhydryl and the disulfide bonds contents), functional (water and oil holding capacities) and secondary and tertiary structure properties. The results showed that the modification effect of PDGs was significantly affected by different extrusion material moisture. The damage degree of the natural structure of PDGs was increased gradually with the increase of extrusion material moisture, and the change of molecular conformation promoted the exposure of internal hydrophilic groups and the formation of disulphide bonds. The solubility was increased by 1.56 times to 34.75%, the surface hydrophobicity was reduced by 56.11% at 27% extrusion materials moisture, and the water holding capacity was enhanced accordingly. The thermal denaturation temperature was increased from 96.79 ℃ to 102.53 ℃, indicating the thermal stability was stronger. The order of protein aggregates increased after the denaturation rearrangement of extruded PDGs. Extrusion led to excessive degeneration of PDGs when the material moisture was too high (higher than 27%), and the destruction of the stable aggregation state would weaken the hydrophilicity, thermal property and order degree of PDGs, indicating that the extrusion material moisture played a very important role in regulating the variation of PDGs properties.