A-type carrier (ATC) proteins are proposed to function in the biogenesis of Fe-S clusters, although their exact role remains controversial. The genome ofMycobacterium smegmatisencodes a single ATC protein, MSMEG_4272, which belongs to the HesB/YadR/YfhF family of proteins. Attempts to generate anMSMEG_4272deletion mutant by two-step allelic exchange were unsuccessful, suggesting that the gene is essential for in vitro growth. CRISPRi-mediated transcriptional knock-down ofMSMEG_4272resulted in a growth defect under standard culture conditions, which was exacerbated in mineral-defined media. The knockdown strain displayed reduced intracellular iron levels under iron-replete conditions and increased susceptibility to clofazimine, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and isoniazid, while the activity of the Fe-S containing enzymes, succinate dehydrogenase, and aconitase were not affected. This study suggests that MSMEG_4272 plays a role in the regulation of intracellular iron levels and is required for in vitro growth ofM. smegmatis, particularly during exponential growth.