This study investigated the effectiveness of mechanical refining to overcome the biomass recalcitrance barrier. Laboratory scale refining was conducted via PFI mill and valley beater refiners using green liquor and Kraft hardwood pulps. A strong positive correlation was determined between sugar recovery and water retention value. Refining produced significant improvements in enzymatic hydrolysis yield relative to unrefined substrates (e.g., sugar recovery increase from 67% to 90%, for 15% lignin Kraft pulp). A maximum absolute enzymatic hydrolysis improvement with refining was observed at enzymatic hydrolysis conditions that produced intermediate conversion levels. For a 91% target sugar conversion, PFI refining at 4000 revolutions allowed for a 32% reduction in enzyme charge for 15% lignin content hardwood Kraft pulp and 96 h hydrolysis time, compared to the unrefined material. Published by Elsevier Ltd.