Backgrounds: Escherichia coli is a Gram-negative bacterium commonly found in human and animal intestinal commensal, mainly parasitic in the large intestine. Intestinal pathogenic E. coli causes a wide variety of severe infectious diseases in humans and animals, such as urinary tract infections, meningitis, peritonitis, septicemia, bacterial pneumonia and neonatal humans and diarrhea weanling piglets. Moreover, its remarkable ability to acquire resistance against most of commercially available antibiotics has led to a global threat to human health. Recent studies indicated that antimicrobial peptides (AMPs) may serve as a promising solution to combat multidrug-resistance microbial infections. In our previous studies, we have designed and synthesized a series of novel cationic AMPs with high antibacterial activity and selectivity against a broad spectrum of Gram-positive and Gram-negative bacteria. Materials & Methods: In the current study, we seek to evaluate their potency against wild-type (wt) and several multidrug-resistant (MDR) strains of E. coli. Proteomic approaches were applied to reveal the differences in protein profiles between wt and MDR strains. MIC analysis was performed to evaluate the antibacterial efficacy of these AMPs against wt and MDR strains. In addition, subproteome analyses were performed to identify important outer and inner membrane proteins involved in AMP-bacteria interactions. Results: Antibacterial activity analysis indicated that all our AMPs were effective against both wt and MDR E. coli strains, with GW-H1-a exhibiting the lowest MIC values, which are 8, 4, and 2 ug/mL against wt, low-resistant strain (1R) and high-resistant strain (8R), respectively. Two-dimensional gel electrophoresis was performed as triplicates in three independent experiments. Image analysis revealed several protein spots significantly altered among wt and MDR strains. These protein spots have been in-gel digested by trypsin and then subjected to LC-ESI-Q-TOF MS/MS analysis. Conclusion: Proteins identified could help delineating the interaction between AMPs and pathogens. Lipopolysaccharide (LPS) competition assay will be performed to identify the reaction target of AMPs on the bacterial surface. These findings would provide support for future application of these novel AMPs as potential therapeutic agents for treatment of infections by MDR bacterial strains.