A new class of antibiotics has been identified that targets the lipopolysaccharide (LPS) transport machinery in Gram-negative bacteria, particularly Acinetobacter. These antibiotics, known as macrocyclic peptides, inhibit LPS transport by binding to the LPS transporter, specifically the LptB2FG complex. The mechanism involves trapping a substrate-bound conformation of the transporter, which stalls the transport process. Structural, biochemical, and genetic studies reveal that these antibiotics recognize a composite binding site involving both the Lpt transporter and its LPS substrate. This discovery identifies an unusual mechanism of lipid transport inhibition and reveals a druggable conformation of the Lpt transporter, providing a foundation for developing new antibiotics against Gram-negative pathogens. The outer membrane of Gram-negative bacteria is an asymmetric bilayer containing phospholipids in the inner leaflet and LPS in the outer leaflet. LPS biosynthesis occurs in the inner membrane, and the molecule must be extracted, transported across the periplasm, and delivered to the cell surface. The LptB2FG complex, along with other proteins, facilitates this transport. The macrocyclic peptides bind to the LptB2FG complex in the presence of LPS, forming a stable complex that prevents LPS transport. Structural studies show that the peptides form extensive contacts with both the transporter and LPS, highlighting a unique mode of inhibition. The binding of these antibiotics is critical for inhibiting the growth of Acinetobacter strains. Mutations in the LptFG proteins reduce susceptibility to the antibiotics, confirming the importance of these interactions. The LPS transport process is also influenced by the LpxM enzyme, which modifies the LPS acyl chain. Loss of LpxM function leads to reduced susceptibility to the antibiotics, indicating the importance of LPS structure in drug binding. The LptC helix movement is essential for the binding of the antibiotics and the transport of LPS. Structural studies show that the LptC helix moves between two states, which is important for coordinating LPS transport with the ATP hydrolysis cycle. The macrocyclic peptides bind to the LptB2FG complex in an intermediate transport state, where LPS is bound within the transporter. This binding prevents the transport of LPS to the outer membrane, leading to cell death. The study also highlights the species-selectivity of these antibiotics, as they are effective against Acinetobacter but not other Gram-negative bacteria. This selectivity is due to differences in the Lpt proteins between species. The structures of the LptB2FG complex in Acinetobacter and E. coli show distinct binding pockets, explaining the species-specific drug susceptibility. The findings provide a roadmap for developing new antibiotics that target the LPS transport machinery in Gram-negative bacteria.A new class of antibiotics has been identified that targets the lipopolysaccharide (LPS) transport machinery in Gram-negative bacteria, particularly Acinetobacter. These antibiotics, known as macrocyclic peptides, inhibit LPS transport by binding to the LPS transporter, specifically the LptB2FG complex. The mechanism involves trapping a substrate-bound conformation of the transporter, which stalls the transport process. Structural, biochemical, and genetic studies reveal that these antibiotics recognize a composite binding site involving both the Lpt transporter and its LPS substrate. This discovery identifies an unusual mechanism of lipid transport inhibition and reveals a druggable conformation of the Lpt transporter, providing a foundation for developing new antibiotics against Gram-negative pathogens. The outer membrane of Gram-negative bacteria is an asymmetric bilayer containing phospholipids in the inner leaflet and LPS in the outer leaflet. LPS biosynthesis occurs in the inner membrane, and the molecule must be extracted, transported across the periplasm, and delivered to the cell surface. The LptB2FG complex, along with other proteins, facilitates this transport. The macrocyclic peptides bind to the LptB2FG complex in the presence of LPS, forming a stable complex that prevents LPS transport. Structural studies show that the peptides form extensive contacts with both the transporter and LPS, highlighting a unique mode of inhibition. The binding of these antibiotics is critical for inhibiting the growth of Acinetobacter strains. Mutations in the LptFG proteins reduce susceptibility to the antibiotics, confirming the importance of these interactions. The LPS transport process is also influenced by the LpxM enzyme, which modifies the LPS acyl chain. Loss of LpxM function leads to reduced susceptibility to the antibiotics, indicating the importance of LPS structure in drug binding. The LptC helix movement is essential for the binding of the antibiotics and the transport of LPS. Structural studies show that the LptC helix moves between two states, which is important for coordinating LPS transport with the ATP hydrolysis cycle. The macrocyclic peptides bind to the LptB2FG complex in an intermediate transport state, where LPS is bound within the transporter. This binding prevents the transport of LPS to the outer membrane, leading to cell death. The study also highlights the species-selectivity of these antibiotics, as they are effective against Acinetobacter but not other Gram-negative bacteria. This selectivity is due to differences in the Lpt proteins between species. The structures of the LptB2FG complex in Acinetobacter and E. coli show distinct binding pockets, explaining the species-specific drug susceptibility. The findings provide a roadmap for developing new antibiotics that target the LPS transport machinery in Gram-negative bacteria.