Biofuels derived from cellulosic biomass offer an alternative to petroleum-based liquid transportation fuels. In order to convert cellulosic biomass into biofuels, size reduction is a necessary step along with pretreatment, enzymatic hydrolysis, and fermentation. In the literature, there are inconsistent reports about why size reduction affects sugar yield (proportional to biofuel yield). An important reason for the inconsistence is that particle formation in current size reduction methods is not well controlled, causing effects of some biomass structural parameters confounded. In this study, a metal-cutting (milling) process is used for size reduction of poplar wood, where particle formation can be well controlled to prevent the effects of multiple parameters from being confounded. The results of this study provide explanations for some inconsistent reports in the literature. These results also reveal some opportunities for future research to understand the effects of size reduction on cellulosic biofuel manufacturing.