Aims: To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv. Methods and Results: The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K-m and V-max values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined. Conclusions: Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K-cat and specific activity of the enzyme were 8.49 s(-1) and 13.4 mumol min(-1) mug(-1) respectively. Significance and Impact of the Study: The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB.