The crystal structures of β-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-α-d-glucopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→4)-d-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93–97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 Å away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, α-EPG and α-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the β-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.