Pyridoxal kinase (PdxK, EC 22.214.171.124) is an important enzyme of vitamin B6 salvage pathway which is required for phosphorylation of B6 vitamers. In the present study, pyridoxal kinase (pdxK) gene from Leishmania donovani (LdPdxK) was cloned and a 33 kDa protein was expressed and kinetically characterized. Site-directed mutagenesis was performed to determine the functional significance of conserved GXGD motif. Mutation of Thr229 to Ala did not affect the catalytic function of LdPdxK however Gly228, Gly230 and Asp231 were found to be indispensible for enzyme activity. To determine the role of LdPdxK in Leishmania promastigotes, LdPdxK overexpressing parasites were generated by episomal expression of the enzyme. The overexpression studies revealed the role of this enzyme in growth and infection of the parasite. In silico analysis of the human and parasite PdxK structure revealed significant differences in the active site region thus highlighting its potential as an antileishmanial drug target. Homology model of LdPdxK was built and was subjected to molecular dynamics simulations. Based on the above information, a pharmacophore was developed and shape based virtual screening was performed to identify potential and selective inhibitors against this essential enzyme. The current data suggests that LdPdxK could be a promising antileishmanial drug target.