Human cytosolic beta-glucosidase, also known as klotho-related protein (KLrP, GBA3), is an enzyme that hydrolyzes various beta-D-glucosides, including glucosylceramide. We recently reported the crystal structure of KLrP in complex with glucose [Y. Hayashi, N. Okino, Y. Kakuta, T. Shikanai, M. Tani, H. Narimatsu, M. Ito, Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase, J. Biol. Chem. 282 (2007) 30889-30900]. Here, we report the crystal structure of a covalent intermediate of the KLrP mutant E165Q, in which glucose was covalently bound to a nucleophile, Glu(373). The structure confirms the double displacement mechanism of the retaining beta-glucosidase. In addition, the structure suggests that a water molecule could be involved in the stabilization of transition states through a sugar, 2-hydroxyl.