Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using in vivo radiolabeling, mass spectrometry, and immunostaining analyses that targeted deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH strain. Furthermore, we analyzed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a two-fold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of M. tuberculosis cell wall components.