In Vibrio cholerae, the second messenger bis-(3′−5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) increases exopolysaccharides production and biofilm formation and decreases virulence and motility. As such, c-di-GMP is considered an important player in the transition from the host to persistence in the environment. c-di-GMP level is regulated through a complex network of more than 60 chromosomal genes encoding predicted diguanylate cyclases (DGCs) and phosphodiesterases. Herein we report the characterization of two additional DGCs, DgcK and DgcL, encoded by integrating conjugative elements (ICEs) belonging to the SXT/R391 family. SXT/R391 ICEs are self-transmissible mobile elements that are widespread among vibrios and several species of enterobacteria. We found that deletion of dgcL increases the motility of V. cholerae, that overexpression of DgcK or DgcL modulates gene expression, biofilm formation and bacterial motility, and that a single amino acid change in the active site of either enzyme abolishes these phenotypes. We also show that DgcK and DgcL are able to synthesize c-di-GMP in vitro from GTP. DgcK was found to co-purify with non-covalently bound flavin mononucleotide (FMN). DgcL's enzymatic activity was augmented upon phosphorylation of its phosphorylatable response-regulator domain suggesting that DgcL is part of a two-component signal transduction system. Interestingly, we found orthologues of dgcK and dgcL in several SXT/R391 ICEs from two species of Vibrio originating from Asia, Africa and Central America. We propose that besides conferring usual antibiotic resistances, dgcKL-bearing SXT/R391 ICEs could enhance the survival of vibrios in aquatic environments by increasing c-di-GMP level.