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Vibrio cholerae is an important human pathogen that causes the diarrheal disease cholera. Colonization of the human host is dependent upon coordinated expression of several virulence factors in response to as yet unknown environmental cues. Bile acids have been implicated in the in vitro regulation of several V. cholerae genes, including those involved in motility, chemotaxis, outer membrane protein production, and virulence factor production. Bile is toxic to bacteria and colonization of the intestinal tract is dependent upon bacterial resistance to bile acids. We have identified and characterized two bile-regulated RND-family efflux systems, named here vexAB and vexCD, that are involved in V. cholerae bile resistance. Mutational analysis revealed that the vexAB system is responsible for in vitro intrinsic resistance of V. cholerae to multiple antimicrobial compounds, including bile acids. In contrast, the vexCD efflux system was specific for certain bile acids and detergents and functioned in conjunction with the vexAB system to provide V. cholerae with high-level bile resistance. Mutants containing deletion of vexB, vexD, and vexB-vexD were able to efficiently colonize the infant mouse suggesting that these efflux systems were dispensable for V. cholerae growth in the small intestines of infant mice.