The relationship between the physicochemical properties of lignocellulosic substrates and enzyme digestion is still not well known. After different pretreatments, cellulose hydrolysis and measurements of physicochemical characteristics by column solute exclusion, particle size analysis, X-ray diffraction, Fourier transform infrared spectroscopy and solid state C-13 nuclear magnetic resonance were performed in this study. Partial least squares was then applied to seek the key factors limiting the rate and extent of cellulose digestion. According to the PLS results, the most important factor for cellulose digestion was accessible interior surface area, followed by delignification and the destruction of the hydrogen bonds. The cellulose digestion at 2 and 24 hr were improved with the increased accessibility of interior surface area to the reporter molecules of 5.1-nm diameter. Removal of lignin and breaking of hydrogen bonds were also found to significantly promote cellulose conversion. Other properties, including the breakdown of intramolecular hydrogen bonds, cellulose crystallinity, and hemicellulose content, had less effect on the efficiency of enzymatic hydrolysis. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 384-392, 2010