The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity toE. coliLptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity.Acinetobacter baumannii(A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden.A. baumanniiLptA shares 28% sequence identity withE. coliLptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 µg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE-optimized derivatives can bind to LptA ofA. baumanniiin vitro, despite the high MIC values. Herein, we present a high-resolution structure ofA. baumanniiLptAm in complex with a thanatin derivative7and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive againstA. baumanniiLptA, despite binding events in vitro.