Cottonseed protein has shown great potential as a biodegradable and renewable resource for industrial processes such as the manufacture of wood adhesives. To improve the recovery of the protein from cottonseed flour, we tested the effects of vigorous blending on the extraction efficiency and recovery yield of one-and two-step procedures for isolation of cottonseed protein. For comparison, the effects on one-step soy protein isolation were also examined. Our data indicated that vigorous blending improved the protein recovery from cottonseed and soy flour as much as 40-60%, compared to mild agitation in the extraction phase. The improvement was likely due to the enhanced solid (flour)-liquid (extracting solvent) interaction, and the increased extraction temperature of the vigorous blending process. Similarities in the protein content, molecular mass distribution pattern, and secondary structure of each type of protein isolates processed under different blending treatments indicated that quality of the isolates was not altered by vigorous blending. However, dissimilarities in molecular mass distribution patterns and secondary structures were identified between the different types of isolates (i. e. total, water soluble, and alkali soluble cottonseed proteins, and total soy protein). These differences will enable us to explore in future work the correlations between cottonseed protein structures and industrial use characteristics (such as adhesive properties).