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Vibrio cholerae's capacity to cause outbreaks of cholera is linked to its survival and adaptability to changes in aquatic environments. One of the environmental conditions that can vary inV. cholerae's natural aquatic habitats is calcium (Ca+2). In this study, we investigated the response ofV. choleraeto changes in extracellular Ca2+ levels. Whole-genome expression profiling revealed that Ca2+ decreased the expression of genes required for biofilm matrix production. Luria–Bertani (LB) medium supplemented with Ca2+ (LBCa2+) causedV. choleraeto form biofilms with decreased thickness and increased roughness, as compared with biofilms formed in LB. Furthermore, addition of Ca2+ led to dissolution in biofilms. Transcription of two genes encoding a two-component regulatory system pair, now termedcalcium-regulatedsensor (carS) andregulator (carR), was decreased in cells grown in LBCa2+. Analysis of null and overexpression alleles ofcarSandcarRrevealed that expression ofvps(Vibriopolysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system. Through epistasis analysis we determined that CarR acts in parallel with HapR, the negative regulator ofvpsgene expression.