A series of thiazolopyrimidine derivatives was designed, synthesized and screened for in-vitro inhibition of Dipeptidyl Peptidase IV (DPP IV). The SAR study indicated the influence of substituted chemical modifications on thiazolopyrimidine scaffold. Compound 9 (IC50 = 0.489 μm) and 10 (IC50 = 0.329 μm) having heterocyclic-substituted piperazine with acetamide linker resulted as most potent DPP IV inhibitors among all the compounds screened. Single dose (10 mg/kg) of both the compounds 9 and 10 significantly reduced glucose excursion during oral glucose tolerance test in streptozotocin induced diabetic rat model. Molecular docking studies illustrated the probable binding mode and interactions of thiazolopyrimidine nucleus and its derivatives at binding site of receptor. The binding site for DPP IV is composed of active site region (catalytic triad of Ser630, Asp708 and His740) including S1 and S2 sub-pocket. The aryl moiety of compounds 9, 10 and 11 were observed to occupy S2 binding pocket and interacted with aromatic ring of Tyr662 and Tyr666 acquired through π-π interaction. Thus, it is indicated that occupancy of the highly hydrophobic S2 pocket is more important for DPP IV inhibitory activity. The present study on substituted thiazolopyrimidine derivatives shows good to moderate inhibitory potential of DPP IV enzyme.