Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) colonization of the gastrointestinal tract is critically dependent on its ability to sense and respond to the external environment. This research aims to evaluate the contribution of bile salts- and ferric iron-induced resistance in EHEC to cationic antimicrobial peptide (CAMP) and the roles of pmrAB and am operon in these events. Results showed that EHEC, treatment with either bile salts or ferric iron induced a dose-dependent resistance to Polymyxin B. This resistance phenotype was lost in each of the pmrA and pmrB mutants. PMB resistance in EHEC was also dependent on the concentration of magnesium and on pH, suggesting the involvement of another two component system, PhoPQ. Mutagenesis of the iron-binding site of PmrB abrogated the induced resistance phenotype. The results of this study provide novel insights critical for our understanding of the molecular basis of pathogenesis and may provide new insights toward prevention strategies.