In Gram-negative bacteria, the outer membrane constitutes the first line of defence against toxic compounds including antibiotics. Lipoproteins are vital structural elements of this barrier and crucial components of machineries underpinning its generation and maintenance. Proper localisation of outer membrane lipoproteins is essential for bacterial survival and requires the dedicated trafficking system LolABCDE. In the terminal stage of transport, LolB, the outer membrane receptor, receives lipoproteins from the periplasmic chaperone LolA and delivers them into the membrane. The essentiality and outer membrane location of LolB makes it an appealing target for the design of new antibiotics. However, this requires detailed understanding of how LolB performs two critical functions; the acceptance of lipoproteins bound to LolA, and their subsequent insertion into the inner leaflet of the outer membrane. I used structure-led mutagenesis, crystallography, biophysical approaches and functional assays to identify residues and regions of LolB essential for its mechanism. Using a LolA mutant with a higher affinity for LolB, I trapped a stable LolA-LolB intermediate and resolved its structure by X-ray crystallography. The structure reveals how the concave surface of LolA interacts with the convex LolB beta-barrel by means of hydrogen bonds and charge-charge interactions, involving three different regions of LolB. Mutation of key residues mediating the interaction was carried out, and the effect on interaction with LolA and lipoprotein acceptance was measured. I identified single mutations that abolished interaction with LolA, and abrogated lipoprotein transfer by targeting multiple charged residues, underlining their importance in LolA-LolB interaction and the transfer mechanism. LolB is likely to undergo conformational changes to accommodate lipoprotein, but how this occurs or where the lipoprotein binds have not been determined. To address this, I mutated residues located in potential hinge regions, and used a fluorescence-based assay to detect opening of the LolB cavity, before assessing the efficiency of lipoprotein transfer. LolB variant proteins which most efficiently accepted lipoprotein were isolated and screened in crystal trials. Following optimisation, the structure of a LolB variant in complex with a lipoprotein fragment was obtained, revealing how the LolB cavity expands to accommodate the three lipoprotein acyl chains and providing molecular details of the interaction. The final step of lipoprotein transport, the insertion activity of LolB, was dissected by developing an in vitro insertion assay using lipid coated silica beads in combination with mutagenesis of LolB residues. These efforts underlined the importance of a key loop and allied with my previous structural data, suggests models of how protein insertion can be achieved. Altogether the combination of structural, biochemical and biophysical approaches provides essential insight into all facets of LolB function. The data presented not only leads to a greater understanding of a fundamental bacterial transport system, but may also assist the rational design of new antibacterial molecules targeting LolB function.