Abstract Key objective of this work was to evaluate the use of cellulosic fraction from sugarcane straw pretreated by liquid hot water (LHW) for butanol production via acetone-butanol-ethanol (ABE) fermentation. Separated hydrolysis and fermentation (SHF), and pre-saccharification and simultaneous saccharification and fermentation (PSSF) were investigated at 10 and 15 % w/v biomass loading. For 15 % w/v, the synergistic effect of weak acids and phenolic compounds made the sugarcane straw hydrolysate poorly fermentable. The 10 % w/v solid load was more favorable (∼ 4-fold higher) in both SHF and PSSF strategies with respect to the ABE production, without including a detoxification step. However, PSSF achieved higher ABE titer (10.5 g/L – SHF; 13.5 g/L – PSSF) and productivity (0.09 g/(L.h) – SHF; 0.14 g/(L.h) – PSSF) when compared with SHF. Using best condition (PSSF at 10 % w/v), it would be possible to estimate a yield of 169 L ABE per ton pretreated sugarcane straw (or 84.5 L ABE per ton of raw sugarcane straw), containing 65 L acetone, 95 L butanol, and 9 L ethanol. This result represents a process efficiency of 28 %, based on carbohydrates content in raw material.