Ethambutol (EMB) is an antimycobacterial drug used extensively for the treatment of tuberculosis caused by Mycobacterium tuberculosis. EMB targets the biosynthesis of the cell wall, inhibiting the synthesis of both arabinogalactan and lipoarabinomannan (LAM), and is assumed to act via inhibition of three arabinosyltransferases: EmbA, EmbB, and EmbC. EmbA and EmbB are required for the synthesis of arabinogalactan, and at least one enzyme (M. tuberculosis EmbA [EmbA(Mt)]) is essential in M. tuberculosis. EmbC(Mt) is also essential for the viability of M. tuberculosis but is involved in the synthesis of LAM. We show that mutations in EmbC(Mt) that reduce its arabinosyltransferase activity result in increased sensitivity to EMB and the production of smaller LAM species in M. tuberculosis. Overexpression of EmbC(Mt) was not tolerated in M. tuberculosis, but overexpression of Mycobacterium smegmatis EmbC (EmbC(Ms)) led to EMB resistance and the production of larger LAM species in M. tuberculosis. Treatment of wild-type M. tuberculosis strains with EMB led to inhibition of LAM synthesis, resulting in the production of smaller species of LAM. In contrast, no change in LAM production was seen in EMB-resistant strains. Overexpression of EmbB(Ms) in M. tuberculosis also resulted in EMB resistance, but at a lower level than that caused by EmbC(Ms). Overexpression of EmbA(Mt) in M. tuberculosis had no effect on EMB resistance. Thus, there is a direct correlation between EmbC activity and EMB resistance, as well as between EmbC activity and the size of the LAM species produced, confirming that EmbC is one of the cellular targets of EMB action.