The facultative human pathogen,Vibrio cholerae, employs two-component signal transduction systems (TCS) to sense and respond to environmental signals encountered during its infection cycle. TCSs consist of a sensor histidine kinase (HK) and a response regulator (RR); theV.choleraegenome encodes 43 HKs and 49 RRs, of which 25 are predicted to be cognate pairs. Using deletion mutants of each HK gene, we analyzed the transcription ofvpsL, a biofilm gene required forVibriopolysaccharide and biofilm formation. We found that aV.choleraeTCS that had not been studied before, now termed Rvv, controls biofilm gene transcription. The Rvv TCS is part of a three-gene operon that is present in 30% of Vibrionales species. Thervvoperon encodes RvvA, the HK; RvvB, the cognate RR; and RvvC, a protein of unknown function. Deletion ofrvvAincreased transcription of biofilm genes and altered biofilm formation, while deletion ofrvvBorrvvClead to no changes in biofilm gene transcription. The phenotypes observed in ΔrvvAdepend on RvvB. Mutating RvvB to mimic constitutively active and inactive versions of the RR only impacted phenotypes in the ΔrvvAgenetic background. Mutating the conserved residue required for kinase activity in RvvA did not affect phenotypes, whereas mutation of the conserved residue required for phosphatase activity mimicked the phenotype of thervvAmutant. Furthermore, ΔrvvAdisplayed a significant colonization defect which was dependent on RvvB and RvvB phosphorylation state, but not on VPS production. We found that RvvA’s phosphatase activity regulates biofilm gene transcription, biofilm formation, and colonization phenotypes. This is the first systematic analysis of the role ofV.choleraeHKs in biofilm gene transcription and resulted in the identification of a new regulator of biofilm formation and virulence, advancing our understanding of the role TCSs play in regulating these critical cellular processes inV.cholerae.