Vibrio vulnificus has been shown to require a global transcription factor, NtrC for mature biofilm development via controlling the biosyntheses of lipopolysaccharide and exopolysaccharide (EPS). Biofilm formation and EPS production were dramatically increased in a medium including a tricarboxylic acid cycle-intermediate as a carbon source. These phenotypes required functional NtrC and were abolished by the addition of ammonium chloride. During the initial stage of biofilm formation, both expression of the ntrC gene and the cellular content of NtrC protein increased. Thus, the regulatory roles of NtrC in EPS biosynthesis were studied with three gene clusters for EPS biosyntheses. Transcriptions of the three clusters were positively controlled by NtrC and showed maximal expression at the early stage of biofilm development. Mutants deficient in one of the genes (VV1_2661, VV2_1579 and VV1_2305) in each cluster showed decreased production of EPS, attenuated ability to form biofilm and lowered cytoadherence to human epithelial cells. However, mutations in VV2_1579 and VV1_2305 resulted in lower cytotoxicity to human cells and mortality to mice than the mutation in VV1_2661. These results demonstrate that NtrC-regulated EPS are crucial in biofilm formation of V. vulnificus, and some EPS components play important roles in interacting with hosts.