Enterohemorrhagic Escherichia coli (EHEC) causes severe food and water-borne illness associated with diarrhea, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (HUS). Previously, we reported that treatment of EHEC with a physiologically relevant bile salt mixture (BSM) upregulates genes encoding a two-component system (TCS) (basRS) and a lipid A modification pathway (arnBCADTEF). The current study examines the effect of BSM treatment on EHEC resistance to human cationic antimicrobials, human defensin, HD-5 and cathelicidin, LL-37. Results show a significant increase in resistance to HD-5 when EHEC are pre-treated with BSM as compared to untreated EHEC. The BS-induced resistance phenotype is lost in each of the arnT and basS mutants. Interestingly, BSM treatment does not affect resistance to LL-37. The results of this study provide evidence that BS serve as an environmental cue by triggering changes via a TCS that result in protective modifications of the bacterial outer membrane, thereby increasing resistance to HD-5.