A mild alkaline pretreatment (deacetylation) prior to the dilute acid pretreatment was evaluated as a strategy to improve the sugars recovery and ethanol production from both hemicellulose and cellulose fractions of rice straw. This pretreatment was carried out using different conditions of temperature (50-70°C) and NaOH loading (20-80mg NaOH/g biomass), which were combined according to a 22 central composite design. In this step the removal of acetyl groups as well as the impact of this step on biomass composition were evaluated. In order to assess the impact of the deacetylation on hemicellulosic hydrolysate composition, the influence of the reaction time (30-90min) and sulfuric acid concentration (0.5-1.5% w/v) was also studied, using the alkaline-pretreated solid (deacetylated) and rice straw in natura. The best sequential pretreatment conditions were scaled-up to 50-L reactor, being obtained a cellulose-rich pretreated solid (cellulignin) and a hemicellulosic hydrolysate, which was concentrated to 70g/L xylose to be used as fermentation medium. A significant improvement on ethanol production from xylose by Scheffersomyces stipitis NRRL Y-7124 was observed when the biomass was submitted to deacetylation (about 4-fold). The enzymatic conversion of cellulose was also improved (from 73 to 89%) when the deacetylated cellulignin was used, resulting in an enhancement of the ethanol production (from 12.7 to 20.4g/L) during the simultaneous saccharification and fermentation with Kluyveromyces marxianus NRRL Y-6860. In brief, biomass deacetylation prior to dilute acid pretreatment was an efficient strategy for rice straw processing, substantially improving the ethanol production from both pentose and hexose sugars.@en