Mycobacterium tuberculosis genome contains eleven serine/threonine protein kinases (STPKs). Among these ST- PKs, PknA is a key component of signal transduction pathway that regulates cell shape and possibly cell division in M. tuberculosis via reversible phosphorylation of intracellular proteins. The in vitro peptide library screen showed that Wag31, a putative cell division protein, was a new substrate phosphorylated by PknA. The signal transduction pathway involving Wag31 and PknA plays a unique role in M. tuberculosis growth regulation that may participate in the pathogenesis of tuberculosis. In this study, genes of PknA, wild-type Wag31 (Wag31WT), phosphoablative Wag31T73A, and phosphomimetic Wag31T73E were cloned and expressed. Far-western analyses were performed using partial purified PknA and completely purified Wag31 proteins (Wag31WT, Wag31T73A, and Wag31T73E). Far-western analysis data revealed that the direct interaction between PknA and Wag31 is dependent on the phos- phorylation state of Wag31, which can represent a novel target for the development of new anti-tuberculosis drugs.