The increasing prevalence of antibiotic-resistant Gram-negative bacteria necessitates the development of novel antimicrobial agents with targeted specificity. In this study, we designed and optimized derivatives of the antimicrobial peptide AA16, which truncated from CD14 protein α-helical region, to selectively target Gram-negative bacteria by enhancing lipopolysaccharide (LPS)-enriched interactions, thereby achieving antibacterial spectrum conversion. Starting from the parent peptide AA16 (Ac-AARIPSRILFGALRVL-Amide), we performed strategic amino acid substitutions based on structure-activity relationship analysis. This led to the identification of AA16-10R, a derivative with a specific substitution at position 10, which demonstrated significantly enhanced antibacterial activity against Gram-negative strains such as Escherichia coli and Pseudomonas aeruginosa, while maintaining low hemolytic activity. Mechanistic studies revealed that AA16-10R exhibited a strong binding affinity to LPS (Kd = 0.15 μM), and its interaction with LPS induced the formation of an α-helical structure. This conformational change facilitated its accumulation on the bacterial outer membrane and disrupted membrane integrity. Our innovative approach of exploiting LPS-enriched interactions successfully converted the antimicrobial spectrum of AA16 derivatives from broad-spectrum to Gram-negative-specific. This study highlights a novel strategy for the rational design of antimicrobial peptides based on specific protein-protein interactions, offering a promising avenue for targeted antimicrobial therapy against Gram-negative pathogens.