Pretreatment is known to make biomass more reactive to cellulase by altering the chemical compositions as well as physical structures of biomass. Simons’ staining technique along with mercury porosimetry was applied on the acid, neutral, and alkaline pretreated materials to measure the accessible surface area of cellulose and pore size distribution of Populus. The results indicated that acid pretreatment is much more effective than water and alkaline pretreatment in terms of cellulose accessibility increase. Further investigation suggests that lignin does not dictate cellulose accessibility to the extent that hemicellulose does, but it does restrict xylan accessibility which in turn controls the access of cellulase to cellulose. The most interesting finding is that severe acid pretreatment significantly decreases the average pore size, i.e. 90% average size decrease could be observed after 60 min dilute acid pretreatment at 160 °C; however, the nano-pore space formed between the coated microfibrils increased after pretreatment, especially with the acid pretreatment, suggesting that this particular type of biomass porosity is probably the most fundamental barrier to effective enzymatic hydrolysis.