The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer that protects the cell from external stressors, such as antibiotics. The Mla transport system is implicated in theMaintenance of OMLipidAsymmetry by mediating retrograde phospholipid transport across the cell envelope. Mla uses a shuttle-like mechanism to move lipids between the MlaFEDB inner membrane complex and the MlaA-OmpF/C OM complex,viaa periplasmic lipid-binding protein, MlaC. MlaC binds to MlaD and MlaA, but the underlying protein–protein interactions that facilitate lipid transfer are not well understood. Here, we take an unbiased deep mutational scanning approach to map the fitness landscape of MlaC fromEscherichia coli, which provides insights into important functional sites. Combining this analysis with AlphaFold2 structure predictions and binding experiments, we map the MlaC-MlaA and MlaC-MlaD protein–protein interfaces. Our results suggest that the MlaD and MlaA binding surfaces on MlaC overlap to a large extent, leading to a model in which MlaC can only bind one of these proteins at a time. Low-resolution cryo-electron microscopy (cryo-EM) maps of MlaC bound to MlaFEDB suggest that at least two MlaC molecules can bind to MlaD at once, in a conformation consistent with AlphaFold2 predictions. These data lead us to a model for MlaC interaction with its binding partners and insights into lipid transfer steps that underlie phospholipid transport between the bacterial inner and OMs.