Mycolic acids (MAs) are a unique class of lipids that are essential for viability, virulence and persistence of Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in MAs biosynthesis represent important class of drug targets. We previously showed that (3R)-hydroxyacyl-ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of keto-MAs and plays a determinant role in Mtb virulence. Here, we discovered that HAD activity requires formation of a tight heterotetramer between HadD and HadB, a HAD unit coded by a distinct chromosomal region. Using biochemical, structural and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. We identified determinants of the ultra-long-chain lipid substrate specificity and revealed details of structure-function relationship. Taken together, our study shows that HadBD, and not HadD, is the biologically relevant functional unit, and these results have important implications for designing innovative antivirulence molecules to fight tuberculosis.