Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens and Mycolic acids are very long C60-C90 α-alkyl β-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from and are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10--epoxystearic acid, and forming a vicinal diol. The results of EphD overproduction in and BCG Δ strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing , we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the gene in specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.