Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositol-based lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 220.127.116.11) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-l-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Analysis of the M. tuberculosis genome suggested the presence of four M. tuberculosis gene products that exhibit an inositol monophosphatase signature. In the present report, we have focused on SuhB, which possesses the highest degree of homology with human inositol monophosphatase. SuhB gene was cloned into an E. coli expression vector to over-produce a His-tagged protein, which was purified and characterized. SuhB required divalent metal ions for functional inositol monophosphatase activity, with Mg2+ being the strongest activator. Inositol monophosphatase activity catalyzed by SuhB was inhibited by the monovalent cation lithium (IC50 = 0.9 mM). As anticipated, inositol-l-phosphate was the preferred substrate (Km = 0.177 ± 0.025 mM; kcat = 3.6 ± 0.2 s-1); however, SuhB was also able to hydrolyze a variety of polyol phosphates such as glucitol-6-phosphate, glycerol-2-phosphate, and 2′-AMP. To provide further insight into the structure-function relationship of SuhB, different mutant proteins were generated (E83D, D104N, D107N, W234L, and D235N). These mutations almost completely abrogated inositol monophosphatase activity, thus underlining the importance of these residues in inositol-l-phosphate dephosphorylation. We also identified L81 as a key residue involved in sensitivity to lithium. The L81A mutation rendered SuhB inositol monophosphatase activity 10-fold more resistant to inhibition by lithium (IC50 = 10 mM). These studies provide the first steps in the delineation of the biosynthesis of the key metabolite inositol in M. tuberculosis.