Bacterial β‐glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as β‐cellulose, β‐glucans and β‐xylans from different sources, which are used in a myriad of biomedical and industrial applications. It has been shown that a conformational change of the substrate, from a relaxed 4C1 conformation to a distorted 1S3/1,4B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate’s distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)‐based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i.e. the one at subsite ‐1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2‐deoxy‐2‐fluoro derivatives, widely used to trap the reaction intermediates by X‐ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2‐OH···nucleophile interaction in substrate recognition and catalysis in endo‐glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.