Lincomycin, produced by the actinomycete Streptomyces lincolnensis, is highly effective against Gram-positive bacteria and protozoans, making it widely used in clinical settings. This study identified LcbR2, a MarR family transcriptional regulator, as an activator of lincomycin biosynthesis. Knocking out the lcbR2 gene reduced lincomycin production by 63.0 % without affecting growth or morphology. Quantitative real-time PCR, electrophoretic mobility shift assays, and XylE reporter assays demonstrated that LcbR2 binds to a 13-bp imperfect palindromic sequence -TTGCCnnnnnCAA-, repressing the expression of lcbR2 Further analysis revealed that LcbR2 directly activates the expression of lincomycin biosynthesis genes (lmbD, lmbJ, lmbK, lmbV, and lmbW), enhancing lincomycin production. It also regulates lincomycin resistance genes (lmrA and lmrB), increasing the self-tolerance of S. lincolnensis to lincomycin. Additionally, LcbR2 modulates other regulatory genes (lmbU, adpA, aflQ1, bldD, and lcbR1), affecting lincomycin production in a cascade manner. LcbR2 also influences the expression of genes related to carbon, nitrogen, phosphorus, and sulfur metabolism, indirectly impacting lincomycin production. Moreover, the binding of LcbR2 to DNA can be attenuated by apramycin. This study thus characterized LcbR2 as a novel transcriptional regulator with a broad regulatory scope.