Acetone-based organosolv fractionation of wheat straw was studied to produce sugars and lignin in a lignocellulose biorefinery. The influence of acetone−water ratio, reaction time, and temperature on fractionation degree, lignin yield, and enzymatic digestibility of the cellulose fraction was examined. The solvent−water ratio was found to influence delignification and hemicellulose hydrolysis by its effect on lignin solubility and pH. Organosolv treatment in 50:50% w/w acetone−water during 1 h at 205 °C resulted in 82% hemicellulose hydrolysis, 79% delignification, and 93% cellulose recovery. A substantial part of the sugars formed upon hemicellulose hydrolysis seems to have reacted further including possible formation of lignin−furfural condensation products. Finally, aqueous acetone pretreatment was found to substantially improve the enzymatic digestibility of the cellulose fraction (glucose yield: up to 87% vs 16% for untreated wheat straw).