Inhibition of glycoside hydrolases has widespread application in treatment of diabetes, viral infections, lysosomal storage diseases and cancers. Gluco-configured tetrahydroimidazopyridines are the most potent β-glucosidase inhibitors reported to date. Using transition state mimic strategy, a series of C2-substituted gluco-configured tetrahydroimidazopyridines were designed and synthesized. Compounds 3 (K i =0.64nM) and 5 (K i =0.58nM) showed stronger inhibitory potency against β-glucosidase. Maestro 9.1 was used to study the structure–activity relationships by docking the compounds into the β-glucosidase active sites.