The survival of Gram-negative bacteria depends on assembly of the asymmetric outer membrane, which creates a barrier that prevents entry of toxic molecules including antibiotics. The outer leaflet of the outer membrane is composed of lipopolysaccharide, which is made at the inner membrane and pushed across a protein bridge that spans the inner and outer membranes. We have developed a fluorescent assay to follow lipopolysaccharide (LPS) transport across a bridge linking proteoliposomes that mimic the inner and outer membranes. We show that LPS is delivered to the leaflet of the outer membrane proteoliposome that corresponds to the outer leaflet of the membrane in a cell. Transport stops long before substrates at the inner membrane are exhausted. Using mutants of the transport machinery, we find that the final amount of LPS delivered into the membrane depends on the affinity of the outer membrane translocon for LPS. Furthermore, ATP hydrolysis depends on delivery of LPS into the outer membrane. Therefore, the transport process is regulated by the outer membrane translocon causing ATP hydrolysis in the inner membrane proteoliposome to stop. Negative feedback from the outer membrane to the inner membrane provides a mechanism for long distance control over LPS transport.