Mycobacterium tuberculosis infects one‐third of the world's population and causes two million deaths annually. The unusually low permeability of its cell wall contributes to the ability of M. tuberculosis to grow within host macrophages, a property required for pathogenesis of infection. Mycobacterium marinum is an established model for discovering genes involved in mycobacterial infection. Mycobacterium marinum mutants with transposon insertions in the β‐ketoacyl‐acyl carrier protein synthase B gene (kasB) grew poorly in macrophages, although growth in vitro was unaffected. Detailed analyses by thin‐layer chromatography, nuclear magnetic resonance (NMR), matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, infrared spectroscopy, and chemical degradations showed that the kasB mutants synthesize mycolic acids that are 2–4 carbons shorter than wild type; the defect was localized to the proximal portion of the meromycolate chain. In addition, these mutants showed a significant (∼30%) reduction in the abundance of keto‐mycolates, with a slight compensatory increase of both α‐ and methoxy‐mycolates. Despite these small changes in mycolate length and composition, the kasB mutants exhibited strikingly altered cell wall permeability, leading to a marked increase in susceptibility to lipophilic antibiotics and the host antimicrobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were fully complemented by expressing M. tuberculosis kasB, but not by the closely related gene kasA. These studies identify kasB as a novel target for therapeutic intervention in mycobacterial diseases.