Biofilms, multicellular microbial communities encased in an extracellular matrix, are a critical factor for bacterial survival and virulence. One positive regulator of biofilm formation is the secondary messenger molecule c-di-GMP which is synthesized by diguanylate cyclases (DGCs), and degraded by phosphodiesterases (PDEs). Vibrio cholerae, the causative agent of the diarrheal disease cholera, has 62 of these genes. One of the critical DGCs for biofilm formation is VpvC which when activated leads to enhanced biofilm formation. However not much is known about this system including how it is regulated. My work identified three transcriptional regulators of the vpv system: the c-di-GMP receptors VpsT and VpsT, and the alternative sigma factor RpoS. These regulators impact global c-di-GMP levels and biofilm formation via vpv regulation. Furthermore, these regulators are critical for vpv expression and 3D architecture in mature biofilms. VpsT and VpsR form a c-di-GMP sensing regulatory circuit for the vpv system while RpoS forms a stationary growth phase regulatory circuit.