Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen. PknI is one of the 11 functional Serine/Threonine Protein Kinases which is predicted to regulate the cell division of M. tuberculosis. In order to find newer drugs and vaccine we need to understand the pathogenesis of the disease. We have used the bioinformatics approach to identify the functionally active residues of PknI and to confirm the same with wet lab experiments. In the current study, we have created homology model for PknI and have done comparative structural analysis of PknI with other kinases. Molecular docking studies were done with a library of kinase inhibitors and T95 was found as the potent inhibitor for PknI. Based on structure based pharmacophore analysis of kinase substrate complexes, Lys 41 along with Asp90, Val92 and Asp96 were identified as functionally important residues. Further, we used site directed mutagenesis technique to mutate Lys 41 to Met resulting in defective cell division of Mycobacterium smegmatis mc2. Overall, the proposed model together with its binding features gained from pharmacophore docking studies helped in identifying ligand inhibitor specific to PknI which was confirmed by laboratory experiments.