Eukaryotic-like Ser/Thr protein kinases (STPKs) are present in many bacterial species, where they control various physiological and virulence processes by enabling microbial adaptation to specific environmental signals. PknJ is the only member of the 11 STPKs identified inMycobacterium tuberculosisthat still awaits characterization. Here we report that PknJ is a functional kinase that forms dimersin vitro, and contains a single transmembrane domain. Using a high-density peptide-chip-based technology, multiple potential mycobacterial targets were identified for PknJ. We confirmed PknJ-dependent phosphorylation of four of these targets: PknJ itself, which autophosphorylates at Thr¹⁶⁸, Thr¹⁷¹and Thr¹⁷³residues; the transcriptional regulator EmbR; the methyltransferase MmaA4/Hma involved in mycolic acid biosynthesis; and the dipeptidase PepE, whose encoding gene is located next topknJin the mycobacterial genome. Our results provide a number of candidate phospho-targets for PknJ and possibly other mycobacterial STPKs that could be studied to investigate the role of STPKs inM. tuberculosisphysiology and virulence.