Nine grain sorghum cultivars with a broad range of ethanol fermentation efficiencies were selected to characterize the changes in sorghum protein in digestibility, solubility, and microstructure during mashing and to relate those changes to ethanol fermentation quality of sorghum. Mashing reduced in vitro protein digestibility considerably, and a large amount of polymers cross-linked by disulfide bonds were developed during mashing. As a marker of cross-linking, protein digestibility of the original samples was highly related to conversion efficiency. gamma-Kafirin (%) neither correlated to ethanol yield nor conversion efficiency significantly. Solubility of proteins in an alkaline borate buffer in conjunction with SDS decreased substantially after mashing. Solubility and the SE-HPLC area of proteins extracted from mashed samples were highly correlated with ethanol fermentation. Ethanol yield increased and conversion efficiency improved notably with the increase of extracted proteins from mashed samples. SE-HPLC total area could be used as an indicator to predict ethanol fermentation. CFLSM images proved that sorghum proteins tended to form highly extended, strong web-like microstructures during mashing. The degree of protein cross-linking differed among samples, and more open microstructures were observed in samples with higher conversion efficiencies. The web-like protein matrix was found to hold not only starch granules but also some oligosaccharides or polysaccharides inside. The formation of web-like microstructures because of cross-linking reduced conversion efficiency.