Plants represent a vast, renewable resource and are well suited to provide sustainably for humankind's transportation fuel needs. To produce infrastructure-compatible fuels from biomass, two challenges remain: overcoming plant cell wall recalcitrance to extract sugar and phenolic intermediates, and reduction of oxygenated intermediates to fuel molecules. To compete with fossil-based fuels, two primary routes to deconstruct cell walls are under development, namely biochemical and thermochemical conversion. Here, we focus on overcoming recalcitrance with biochemical conversion, which uses low-severity thermochemical pretreatment followed by enzymatic hydrolysis to produce soluble sugars. Many challenges remain, including understanding how pretreatments affect the physicochemical nature of heterogeneous cell walls; determination of how enzymes deconstruct the cell wall effectively with the aim of designing superior catalysts; and resolution of issues associated with the co-optimization of pretreatment, enzymatic hydrolysis, and fermentation. Here, we highlight some of the scientific challenges and open questions with a particular focus on problems across multiple length scales.