The drastic increase in ethanol production over the last 10 years has led to overproduction of distiller's grain (DG), an ethanol byproduct. In this study, we investigated the feasibility of extracting the protein from unmilled and milled DG leaving a carbohydrate-rich solid phase that could be used for the production of more ethanol. Protein extractions based on aqueous ethanol (both with and without a pretreatment of a reducing agent), alkaline-ethanol, and aqueous enzyme treatments were compared.;Three methods led to a significant amount of protein being extracted from DG. An enzymatic extraction decreased the crude protein content in the solid phase for both the unmilled and the milled DG from 38% (dry weight) to approximately 8% (dry weight) protein. The alkaline-ethanol extraction was similarly effective for the milled DG but not for the unmilled material. Simple extraction with alcohol, both with and without a reducing agent, was not as effective.