The use of aqueous ionic liquids as a pretreatment for enhancing enzymatic saccharification of lignocellulosic biomass is well-known with focus on lignin and hemicellulose removal. However, an in-depth knowledge of changes in cellulose crystallinity during the pretreatment is limited. To this effect, detailed FT-IR, CP/MAS 13C NMR and XRD studies sugarcane bagasse pretreated with aqueous ionic liquids is presented. Secondary-derivatives of FT-IR spectra and Gauss deconvolution of XRD patterns were applied to give detailed understanding of sugarcane bagasse cellulose crystalline changes upon the pretreatment. The results showed that aqueous ionic liquids pretreatment destroys the semi-crystalline region of sugarcane bagasse cellulose, thus disordering the orientation of cellulose crystallite.