l-Arginine serves as a carbon and nitrogen source and is critical for (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of (), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in bacille Calmette-Guerin.