In recent years the antimicrobial peptides (AMPs) have been prospected and designed as new alternatives to conventional antibiotics. Indeed, AMPs have presented great potential toward pathogenic bacterial strains by means of complex mechanisms of action. However, reports have increasingly emerged regarding the mechanisms by which bacteria resist AMP administration. In this context, we performed a comparative proteomic study by using the total bacterial lysate of magainin I-susceptible and -resistant E. coli strains. After nanoUPLC-MSE analyses we identified 742 proteins distributed among the experimental groups, and 25 proteins were differentially expressed in the resistant strains. Among them 10 proteins involved in bacterial resistance, homeostasis, nutrition and protein transport were upregulated, while 15 proteins related to bacterial surface modifications, genetic information and β-lactams binding-protein were downregulated. Moreover, 60 exclusive proteins were identified in the resistant strains, among which biofilm and cell wall formation and multidrug efflux pump proteins could be observed. Thus, differentially from previous studies that could only associate single proteins to AMP bacterial resistance, data here reported show that several metabolic pathways may be related to E. coli resistance to AMPs, revealing the crucial role of multiple "omics" studies in order to elucidate the global molecular mechanisms involved in this resistance.