In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry. DOI: http://dx.doi.org/10.7554/eLife.19042.001, eLife digest Escherichia coli are bacteria that can cause vomiting and diarrhoea in humans and other mammals. Each E. coli cell is surrounded by two membranes, which are each made of two layers of fat molecules known as lipids. The outer membrane prevents the entry of toxic compounds and allows E. coli to withstand damaging agents from outside the cell, such as antibiotics. The outer membrane’s ability to act as an effective barrier depends on an asymmetric, or uneven, distribution of lipid molecules across its two layers. The inside layer is dominated by phospholipids, whereas the outside layer is comprised mainly of lipids with attached sugars. The distribution of the two lipid types is maintained by a molecular machine with components that can be found in both the inner and outer membranes. This machine is thought to remove phospholipids from the outside layer of the outer membrane and transport them back to the inner membrane. A group (or”complex”) of proteins known as MIaFEDB operates as a part of this machine at the inner membrane. MlaFEDB is believed to use energy derived from the breakdown of a molecule called ATP to help ensure that phospholipids removed from the outside layer of the outer membrane are reinserted into the inner membrane. It was proposed that the complex contains four proteins, but it was not clear exactly how these components are arranged. Now, Thong et al. reveal how MlaFEDB is organized and characterize the roles of the individual protein components. The experiments confirm that the MlaFEDB complex is made up of four proteins, including two core components and two support proteins (called MlaB and MlaD). There are six copies of MlaD in the complex. In addition, MlaD has a strong affinity for phospholipids and plays a role in controlling the rate at which energy is harnessed through the breakdown of ATP. Further experiments show that the other support protein MlaB is necessary for both the proper assembly and activity of the complex, likely through its interaction with one of the core components. The next step following on from this work is to directly observe MlaFEDB in action to find out how it uses energy to insert lipids into the inner membrane. In the long term, more information about the structure of the complex would be needed to further understand how it works at the molecular level. DOI: http://dx.doi.org/10.7554/eLife.19042.002