The aim of this study is to convert spent mushroom substrate (SMS), a by-product from industrial mushroom production, to high production of biobutanol via integration with in situ butanol removal strategy. The modified organosolv pretreatment under mild conditions (35 degrees C, without acid catalyst) could efficiently improve the sugar yield (9.7% Vs 41.7%) of SMS by reducing lignin content (20.2% Vs 10.5%). The batch fermentation results using the organosolv treated SMS enzymatic broth indicate that adding extractant of biodiesel could enhance butanol production (14.65 g/L) compared to the control batch (12.03 g/L). Moreover, no significant toxicity of biodiesel is observed on the growth of solvento-genic Clostridium acetobutylicum. In the fed-batch fermentation system with sugar feeding, the maximum amount of butanol is obtained at 30.21 g/L. This study suggests that in situ butanol removal using biodiesel from SMS enzymatic hydrolysate has great potential for commercial butanol production. (C) 2016 Published by Elsevier Ltd.