MmpL3 is a promising target for novel anti-tubercular agents, with numerous compound series identified as MmpL3 inhibitors. Despite this, there is an incomplete understanding of MmpL3 function. Here we show that Mycobacterium smegmatis MmpL3 mutant strains had an altered cell wall hydrophobicity, disrupted membrane potential and growth defects in liquid media. Compensatory mutations that restored normal growth also returned membrane potential to wild-type. M. smegmatis MmpL3 mutant strains were resistant to two anti-tubercular agents, SQ109 and AU1235, but were more sensitive to rifampicin, erythromycin and ampicillin. Exposure of M. smegmatis to AU1235 affected the cell wall composition and increased the potency of rifampicin. However, MmpL3 mutants did not prevent the dissipation of membrane potential following exposure to SQ109. These results demonstrate that in M. smegmatis, MmpL3 contributes to a number of important phenotypes such as membrane potential, cell wall composition, antibiotic susceptibility and fitness.