Objective: The aim of this study was to evaluate the dipeptidyl peptidase-IV (DPP-IV) inhibitor sitagliptin with respect to mode of inhibition and its in vivo duration of inhibition and efficacy in type 2 diabetes animal model. Materials and Methods: DPP-IV enzyme assay was carried out in human plasma (10 μL) or human recombinant enzyme (10 ng) using H-Gly-Pro-AMC as a substrate. The competitive nature was estimated by plotting IC50 values measured at different substrate concentrations on the Y axis and substrate concentration on the X axis. The tight binding nature was estimated by plotting IC50 values measured at different plasma volumes on the Y axis and plasma volumes on the X axis. Fast binding kinetics was assessed by progressive curves at different inhibitor concentrations in the DPP-IV assay. The reversibility of the inhibitor was assessed by a dissociation study of the DPP-IV-sitagliptin complex. Durations of DPP-IV inhibition and efficacy were shown in ob/ob mice dosed at 10 mg/kg, p.o. Results: Sitagliptin is a competitive, reversible, fast and tight binding DPP-IV inhibitor. In ob/ob mice, 10 mg/kg, (p.o.) showed a long duration of inhibition of > 70% at 8 h. The duration was translated into long duration of efficacy (~ 35% glucose excursion at 8 h) in the same model and the effect was comparable to vildagliptin. Conclusion: The DPP-IV inhibitor sitagliptin behaves as a competitive, tight, and fast binding inhibitor. Sitagliptin differs mechanistically from vildagliptin and exhibits comparable efficacy to that of latter. The finding may give an understanding to develop-second generation DPP-IV inhibitors with desired kinetic profiles.