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Summary Tuberculosis claims >1 million lives annually, and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesize peptidoglycan in an osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identify CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM: a non-phosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore, we show that the partner proteins for the phosphorylated and non-phosphorylated forms of CwlM are FhaA, a fork head-associated domain protein, and MurJ, a proposed lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane. Graphical Abstract Highlights • PknB is not critical for M. tuberculosis growth in osmoprotective medium • CwlM is the major substrate of PknB • Phosphorylation controls localization of CwlM in the cytoplasmic and membrane fractions • Phospho-CwlM binds FhaA, and non-phospho-CwlM interacts with the essential MurJ linker PknB controls growth and peptidoglycan biosynthesis in Mycobacterium tuberculosis. Turapov et al. show that CwlM, a major PknB substrate, is produced in two forms: a non-phosphorylated membrane-associated CwlM and a PknB-phosphorylated cytoplasmic CwlM. The phosphorylated CwlM binds FhaA, a fork head-associated domain protein, while non-phosphorylated CwlM interacts with MurJ (MviN), a proposed lipid II flippase. [ABSTRACT FROM AUTHOR]