Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higherk_cat/K_mvalue for 4-nitrophenyl β-D-mannoside (4NPMan) compared with 4-nitrophenyl β-D-glucoside (4NPGlc). To investigate its selectivity for β-D-mannoside and β-D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Å and of Os7BGlu26 with mannose at a resolution of 2.45 Å were elucidated from isomorphous crystals in space groupP2₁2₁2₁. The (β/α)₈-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with D-mannose corresponds to a product complex, with β-D-mannose in the¹S₅skew-boat conformation. Docking of the¹S₃,¹S₅,²S_Oand³S₁pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the¹S₅and¹S₃skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without β-D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similark_cat/K_mvalues for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higherk_cat/K_mfor 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for β-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.