The mycolic acid transporter MmpL3 is driven by proton motive forces (PMF) and functions via an antiport mechanism. Although the crystal structures of the Mycobacterium smegmatis MmpL3 transporter alone and in complex with a trehalose monomycolate (TMM) substrate and an antituberculosis drug candidate SQ109 under Phase 2b-3 Clinical Trials are available, no water and no conformational change in MmpL3 were observed in these structures to explain SQ109's inhibition mechanism of proton and TMM transportation. In this study, molecular dynamics simulations of both apo form and inhibitor-bound MmpL3 in an explicit membrane were used to decipher the inhibition mechanism of SQ109. In the apo system, the close-open motion of the two TM domains, likely driven by the proton translocation, drives the close-open motion of the two PD domains, presumably allowing for TMM translocation. In contrast, in the holo system, the two PD domains are locked in a closed state, and the two TM domains are locked in an off pathway wider open state due to the binding of the inhibitor. Consistent with the close-open motion of the two PD domains, TMM entry size changes in the apo system, likely loading and moving the TMM, but does not vary much in the holo system and probably impair the movement of the TMM. Furthermore, we observed that water molecules passed through the central channel of the MmpL3 transporter to the cytoplasmic side in the apo system but not in the holo system, with a mean passing time of ∼135 ns. Because water wires play an essential role in transporting protons, our findings shed light on the importance of PMF in driving the close-open motion of the two TM domains. Interestingly, the key channel residues involved in water passage display considerable overlap with conserved residues within the MmpL protein family, supporting their critical function role.