A cluster of genes encoded by ORFs Rv0014c-Rv0018c in Mycobacterium tuberculosis encodes candidate cell division proteins RodA and PBPA, a pair of serine/threonine kinases (STPKs), PknA and PknB, and a phosphatase, PstP. The organization of genes encompassing this region is conserved in a large number of mycobacterial species. This study demonstrates that recombinant PBPA of M. tuberculosis binds benzylpenicillin. Knockout of its counterpart in M. smegmatis resulted in hindered growth and defective cell septation. The phenotype of the knockout (PBPA-KO) could be restored to that of the wild-type upon expression of PBPA of M. tuberculosis. PBPA localized to the division site along with newly synthesized peptidoglycan, between segregated nucleoids. In vivo coexpression of PBPA and PknB, in vitro kinase assays and site-specific mutagenesis substantiated the view that PknB phosphorylates PBPA on T362 and T437. A T437A mutant could not complement PBPA-KO. These studies demonstrate for the first time that PBPA, which belongs to a subclass of class B high-molecular-mass PBPs, plays an important role in cell division and cell shape maintenance. Signal transduction mediated by PknB and PstP likely regulates the positioning of this PBP at the septum, thereby regulating septal peptidoglycan biosynthesis.