The enzymatic conversion of cellulose to fermentable sugars is an attractive way to produce renewable feedstocks for fuels and chemicals; however, native cellulose is recalcitrant to enzymatic degradation and must be chemically and/or physically pretreated to improve its susceptibility to enzymatic attack. Previous hydrolysis studies using “model” cellulosic substrates have been unable to completely decouple the effects of various substrate properties such as degree of polymerization, degree of crystallinity, and accessible surface area on hydrolyzability; consequently, pretreatment recipes must be optimized largely by trial-and-error. Here, we electrospin cellulose to produce uniform fibers with carefully controlled and systematically varied microstructure to investigate the effects of various substrate properties on hydrolyzability. Knowledge gleaned from hydrolysis studies with electrospun cellulose has the potential to provide new directions for pretreatment that will optimize the hydrolysis of native cellulose. We present results for the effects of substrate properties on hydrolysis, and we discuss some new insights about the hydrolysis process that have been revealed through the use of electrospun cellulose as a model substrate.