In recent years, various dipeptidyl peptidase IV (DPP-4) inhibitors have been released as therapeutic drugs for type 2 diabetes in many countries. In spite of their diverse chemical structures, no comparative studies of their binding modes in the active site of DPP-4 have been disclosed. We determined the co-crystal structure of vildagliptin with DPP-4 by X-ray crystallography and compared the binding modes of six launched inhibitors in DPP-4. The inhibitors were categorized into three classes on the basis of their binding subsites: (i) vildagliptin and saxagliptin (Class 1) form interactions with the core S-1 and S-2 subsites and a covalent bond with Ser630 in the catalytic triad; (ii) alogliptin and linagliptin (Class 2) form interactions with the S'(1) and/or S'(2) subsites in addition to the S-1 and S-2 subsites; and (iii) sitagliptin and teneligliptin (Class 3) form interactions with the S-1, S-2 and S-2 extensive subsites. The present study revealed that the additional interactions with the S'(1), S'(2) or S-2 extensive subsite may increase DPP-4 inhibition beyond the level afforded by the fundamental interactions with the S-1 and S-2 subsites and are more effective than forming a covalent bond with Ser630. (c) 2013 Elsevier Inc. All rights reserved.