Lipid transporters from the major facilitator superfamily (MFS) have been reported to play central roles in diverse fundamental biological pathways. However, little is known about their transport mechanisms and only the structure of the MFS lipid transporter LtaA in an outward-facing conformation has been determined. LtaA was shown to be a proton-dependent lipid transporter, essential for anchoring of lipoteichoic acids (LTA) to the plasma membrane of the pathogenic bacteria Staphylococcus aureus. Here we used a ‘repeat swap’ approach in combination with cross-linking analysis to predict an inward-facing state of LtaA, performed molecular dynamics simulations, in vitro and in vivo activity assays, and mutagenesis analysis to characterize outward- and inward-facing conformations of LtaA in lipid membranes. Our results indicate that LtaA catalyze lipid translocation by a “trap-and-flip” mechanism, paving the way for the mechanistic characterization of other MFS lipid transporters.