Significance: The increasing development of antimicrobial resistance is a major global concern, and there is an urgent need for the development of new antibiotics. We show that the antimicrobial lipopeptide tridecaptin A₁selectively binds to the Gram-negative analogue of peptidoglycan precursor lipid II, disrupting the proton motive force and killing Gram-negative bacteria. We present an example of the selective targeting of Gram-negative lipid II and a binding mode to this peptidoglycan precursor. No persistent resistance develops against tridecaptin A₁inEscherichia colicells exposed to subinhibitory concentrations of this peptide during a 1-mo period. This study showcases the excellent antibiotic properties of the tridecaptins in an age where new antibiotics that target Gram-negative bacteria are desperately needed.