Pretreatment facilitates more complete deconstruction of plant biomass to enable more economic production of lignocellulosic biofuels and byproducts. Various co-solvent pretreatments have demonstrated advantages relative to aqueous-only methods by enhancing lignin removal to allow unfettered access to cellulose. However, there is a limited mechanistic understanding of the interactions between the co-solvents and cellulose that impedes further improvement of such pretreatment methods. Recently, tetrahydrofuran (THF) has been identified as a highly effective co-solvent for the pretreatment and fractionation of biomass. To elucidate the mechanism of the THF water interactions with cellulose, we pair simulation and experimental data demonstrating that enhanced solubilization of cellulose can be achieved by the THF water co-solvent system at equivolume mixtures and moderate temperatures (<445 K). The simulations show that THF and water spontaneously phase separate on the local surface of a cellulose fiber, owing to hydrogen bonding of water molecules with the hydrophilic cellulose faces and stacking of THF molecules on the hydrophobic faces. Furthermore, a single fully solvated cellulose chain is shown to be preferentially bound by water molecules in the THF water mixture. In light of these findings, co-solvent reactions were performed on microcrystalline cellulose and maple wood to show thatTHF significantly enhanced cellulose deconstruction and lignocellulose solubilization at simulation conditions, enabling a highly versatile and efficient biomass pretreatment and fractionation method.