Avian pathogenic (APEC), an extra-intestinal pathogenic (ExPEC), causes colibacillosis in chickens and is reportedly associated with urinary tract infections and meningitis in humans. Development of resistance is a major limitation of current ExPEC antibiotic therapy. New antibacterials that can circumvent resistance problem such as antimicrobial peptides (AMPs) are critically needed. Here, we evaluated the efficacy of GG (LGG) derived peptides against APEC and uncovered their potential antibacterial targets. Three peptides (NPSRQERR: P1; PDENK: P2, and VHTAPK: P3) displayed inhibitory activity against APEC. These peptides were effective against APEC in biofilm and chicken macrophage HD11 cells. Treatment with these peptides reduced the cecum colonization (0.5 to 1.3 logs) of APEC in chickens. Microbiota analysis revealed two peptides (P1 and P2) decreased Enterobacteriaceae abundance with minimal impact on overall cecal microbiota of chickens. Bacterial cytological profiling showed peptides disrupt APEC membrane either by causing membrane shedding, rupturing or flaccidity. Further, gene expression analysis revealed that peptides downregulated the expression of C (>13.0 folds), F (>11.3 folds) and A (>4.9 folds) genes responsible for maintenance of outer membrane (OM) lipid asymmetry. Consistently, immunoblot analysis also showed decreased levels of OmpC and MlaA proteins in APEC treated with peptides. Alanine scanning studies revealed residues crucial (P1: N, E, R and P; P2: D and E; P3: T, P, and K) for their activity. Overall, our study identified peptides with new antibacterial target that can be developed to control APEC infections in chickens, thereby curtailing poultry-originated human ExPEC infections. APEC is a subgroup of ExPEC and considered as a foodborne zoonotic pathogen transmitted through consumption of contaminated poultry products. APEC shares genetic similarities with human ExPECs, including uropathogenic (UPEC) and neonatal meningitis (NMEC). Our study identified LGG-derived peptides (P1: NPSRQERR, P2: PDENK, and P3: VHTAPK) effective in reducing APEC infection in chickens. Antimicrobial peptides (AMPs) are regarded as ideal candidates for antibacterial development because of their low propensity for resistance development and ability to kill resistant bacteria. Mechanistic studies showed peptides disrupt APEC membrane by affecting MlaA-OmpC/F system responsible for maintenance of OM lipid asymmetry, a promising new druggable target to overcome resistance problem in Gram-negative bacteria. Altogether, these peptides can provide a valuable approach for development of novel anti-ExPEC therapies, including APEC, human ExPECs and other related Gram-negative pathogens. Further, effective control of APEC infections in chickens can curb poultry-originated ExPEC infections in humans.