Significance Our studies show that the mycolic acid desaturase regulator (MadR) acts as a molecular switch, controlling the desaturation and biosynthesis of mycolic acids, key lipids of the cell envelopes of mycobacteria. MadR works by a distinct mechanism wherein it binds various acyl-coenzyme As (aceyl-CoAs), but only saturated acyl-CoAs relieve DNA binding and repression. This suggests a unique mechanism that involves sensing of acyl-CoA pools as a checkpoint for coordinating mycolic acid remodeling and biosynthesis in response to cell surface perturbation. Our findings further our understanding of how mycobacteria control cell wall composition in response to stress across various environments ranging from soil to an intracellular niche in infected macrophages, with implications for understanding strategies for pathogenesis in the tubercle bacillus.