Visceral leishmaniasis is a neglected tropical disease. Drug resistance and toxicity are the critical issues with the currently available antileishmanial drugs. Therefore, research efforts are underway to identify and validate new drug targets specific to Leishmania parasite. The enzyme homoserine dehydrogenase (HSD) functions in the third step of aspartate pathway. The present study focuses on the biophysical and biochemical characterization of HSD enzyme from Leishmania donovani (LdHSD) which is unique to the parasite with no homologous enzyme in the host. LdHSD gene was cloned in pET28c(+) vector and transformed in E. coli BL21 (DE3) strain. LdHSD recombinant enzyme of molecular weight 46.6 kDa with 6X-His tag at the C-terminal end was expressed, purified by nickel affinity chromatography and confirmed by western blot analysis using anti-His antibody. Effect of pH, temperature, salts, metal ions and amino acids on the recombinant enzyme were evaluated. Kinetic parameters of LdHSD were evaluated for substrates L-homoserine and NADP+. Biophysical analysis revealed that the enzyme is rich in β-sheets. Thermal denaturation study revealed that the protein is stable up to 45 °C. Furthermore, comprehensive comparative sequence analysis and structural modeling revealed the structural and functionally important residues, which are involved in the catalytic mechanisms. The putative binding mode of the natural substrate L-homoserine into the active site of LdHSD was also elucidated. These findings provide a foundation for the development of selective, target-based inhibitors against the HSD enzyme of the parasite.