© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr213. The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrientstarved model of persistence and is needed for long term infection of murine macrophages.