The use of aqueous ionic liquids as a pretreatment for enhancing enzymatic saccharification of lignocellulosic biomass is wellknown, with a focus on lignin and hemicellulose removal. However, in-depth knowledge of changes in cellulose crystallinity during the pretreatment is limited. To this effect, detailed Fourier transform infrared (FT-IR) spectroscopy, cross-polarized magic angle spinning 13 C nuclear magnetic resonance (CP/MAS 13 C NMR), and X-ray diffraction (XRD) studies on sugar cane bagasse that has been pretreated with aqueous ionic liquids is presented. Secondary derivatives of FT-IR spectra and Gauss deconvolution of XRD patterns were applied to give a detailed understanding of the crystalline changes in sugar cane bagasse cellulose upon pretreatment. The results showed that aqueous ionic liquid pretreatment destroys the semicrystalline region of sugar cane bagasse cellulose, thus disordering the orientation of the cellulose crystallites.