Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism.