Corn-based ethanol, the most common first generation biofuel in the US, plays an important role as a fossil fuels alternative. Second generation biofuels, which are mostly based on lignocellulosic biomass, have gained great attention in recent years. Size reduction of the lignocellulosic biomass is a key step to the efficiency of downstream processes (i.e., pretreatment, enzymatic hydrolysis, and fermentation), which will affect the yield of ethanol significantly. However, size reduction consumes considerable energy itself, it is an expensive process and needs to be optimized. Some studies have been done on corn stover size reduction, but none of them have examined the initial particle size of feedstock as a variable, and they failed to take the biomass harvest and storage conditions and downstream process requirements into consideration when setting the variables. The objective of this research was to study the effect of initial particle size of corn stover, moisture content, and screen size on energy consumption of corn stover size reduction. Consequently, these results should be generally applicable over a range of conditions that are affected by corn stover harvest and storage conditions and downstream processing.