Enterohemorrhagic Escherichia coli (EHEC) possess the ability to respond to its surroundings though two-component systems. We hypothesized that gastrointestinal cues such as bile, iron and pH promote EHEC resistance to cationic antimicrobial peptides (CAMPs) during infection. Killing assays reveal that exposure to low pH, high iron or bile salt mixture results in increased CAMP resistance that is dependent on pmrB but independent of pmrD. Low Mg+2 -induced CAMP resistance is dependent on pmrD. pmrD promoter analysis indicate that EHEC responds to PhoPQ-inducing conditions by increasing pmrD expression. However, pmrD expression is repressed upon exposure to low acid, bile salt mix and iron. This study suggests that a complex interplay of PhoPQ, PmrAB and PmrD is involved in EHEC’s response to various microenvironmental signals and in the promotion of EHEC’s resistance to CAMPS. The results also provide intriguing evidence of both cooperation and redundancy in the mediation of CAMP resistance by these molecular players.