A single polar flagellum and motility are potential virulence factors of Vibrio vulnificus, a foodborne pathogen. In the present study, the functions of FlhF and regulatory characteristics of the flhF expression of V. vulnificus were investigated. A deletion mutation in flhF abolished motility, flagella formation and flagellin synthesis, and introduction of flhF in trans complemented the defects. The flhF mutant revealed decreased expression of the class III and IV flagella genes, indicating that FlhF is a key regulator for the flagellar biogenesis of V. vulnificus. The influence of global regulatory proteins on the expression of flhF was examined and SmcR, a LuxR homologue, was found to downregulate flhF expression at the transcriptional level. SmcR represses flhF expression only in the stationary phase of growth and exerts its effects by directly binding to the flhF promoter region. Finally, an SmcR binding site, centred at 22.5 bp upstream of the transcription start site, was identified by a DNase I protection assay. The combined results demonstrate that a quorum sensing master regulator SmcR influences the motility and flagellar biogenesis of V. vulnificus through modulating the expression of FlhF in a growth-phase-dependent manner.