Abstract Glycoside hydrolase family 65 (GH65) includes glycoside hydrolases active on various α-glucosides. We previously demonstrated that the GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) is a kojibiose hydrolase and determined its 3-dimensional structure. In this study, the effects of glucosidase inhibitors on FjGH65A and their complex structures were analyzed to elucidate their inhibition mechanism. FjGH65A was competitively inhibited by 1-deoxynojirimycin (DNJ) and noncompetitively inhibited by castanospermine (CSP) with Ki values of 2.95 and 3.69 µm, respectively. The crystal structures of FjGH65A complexed with the inhibitors indicated that DNJ was bound to subsite −1 of FjGH65A, while CSP was bound to subsites −1 and +1 of FjGH65A. Compared with the glucose complex structure, the conformation of Tyr337 was changed in the CSP complex structure. These results provide new structural insights into the mechanism of inhibition against GH65 α-glucoside hydrolases.