The treatment of infections caused by drug-resistant Gram-negative pathogens is challenging as only few viable therapeutic options are available. We hypothesized that conjugating a fatty acid moiety onto a peptide dimer could maximize the interaction with lipopolysaccharide (LPS) and facilitate the permeabilization of the LPS barrier, thereby improving potency against Gram-negative pathogens.[1] This study is to systematically design a series of N-lipidated peptide-dimers (Cn-B2088) that displayed potent antimicrobial activities against Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE). Methods: In vitro anti-microbial activity of the N-lipidated peptides were evaluated using a panel of Gram-negative bacteria, including multi-drug resistant Pseudomonas aeruginosa and CRE. Their interactions with outer and inner membrane of Gram-negative bacteria were also investigated by use of biophysical and biochemical protocols. Results: The optimized lipid length consisted of carbon numbers of 8. C8-B2088 displayed potent and stable antimicrobial activity against all the P. aeruginosa and CRE strains tested (MICs= 3- 10 µM). It also showed rapid time-kill, promising selectivity and low toxicity and active against Gram-negative pathogens. At lipid lengths of n= 6-10, the N-lipidated peptide-dimers showed strong outer membrane permeabilization. Inner membrane disruption was also observed. As C8- and C-10 B2088 showed stronger interaction with LPS, we further investigated the ability of C6- and C8-B2088 to synergize with other antibiotics, including gentamicin, erythromycin, streptomycin and kanamycin. We found that both compounds could achieve fractional inhibitory concentration (FIC) index of 0.25 - 5 against E. coli and P. aeruginosa. Conclusion: This study shows that design of novel molecule based on complementary binding to LPS may be an efficient approach against Gram-negative bacteria. We have identified C8-B2088 as a potential therapeutic agent for Gram-negative bacteria infections. Our design of the N-lipidated peptide-dimers with excellent toxicity profile represents a promising class of peptide candidates in combating the growing concern of increasing bacterial resistance.