The lipid environment in which membrane proteins are embedded can influence their structure and function. Lipid-protein interactions and lipid-induced conformational changes necessary for protein function remain intractable in vivo using high-resolution techniques. Using Escherichia coli strains in which the normal phospholipid composition can be altered or foreign lipids can be introduced, we established the importance of membrane lipid composition for the proper folding, assembly, and function of E. coli lactose (LacY) and sucrose (CscB) permeases. However, the molecular mechanism underlying the lipid dependence for active transport remains unknown. Herein, we demonstrate that the structure and function of CscB and LacY can be modulated by the composition of the lipid environment. Using a combination of assays (transport activity of the substrate, protein topology, folding, and assembly into the membrane), we found that alterations in the membrane lipid composition lead to lipid-dependent structural changes in CscB and LacY. These changes affect the orientation of residues involved in LacY proton translocation and impact the rates of protonation and deprotonation of E325 by affecting the arrangement of transmembrane domains in the vicinity of the R302-E325 charge pair. Furthermore, the structural changes caused by changes in membrane lipid composition can be altered by a single-point mutation, highlighting the adaptability of these transporters to their environment. Altogether, our results demonstrate that direct interactions between a protein and its lipid environment uniquely contribute to membrane protein organization and function. Because members of the major facilitator superfamily present with well-conserved functional architecture, we anticipate that our findings can be extrapolated to other membrane protein transporters.