Receptor Ser/Thr protein kinases are candidates for sensors that govern developmental changes and disease processes of Mycobacterium tuberculosis (Mtb), but the functions of these kinases are not established. Here, we show that Mtb protein kinase (Pkn) D overexpression alters transcription of numerous bacterial genes, including Rv0516c, a putative anti-antisigma factor, and genes regulated by sigma factor F. The PknD kinase domain directly phosphorylated Rv0516c, but no other sigma factor regulator, in vitro. In contrast, the purified PknB and PknE kinase domains phosphorylated distinct sigma regulators. Rather than modifying a consensus site, PknD phosphorylated Rv0516c in vitro and in vivo on Thr2 in a unique N-terminal extension. This phosphorylation inhibited Rv0516c binding in vitro to a homologous anti-antisigma factor, Rv2638. These results support a model in which signals transmitted through PknD alter the transcriptional program of Mtb by stimulating phosphorylation of a sigma factor regulator at an unprecedented control site.