Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers. This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators. Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout. In this study, we sought to determine whether V. anguillarum employs AHLs to regulate virulence gene expression. Spent V. anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum. This finding suggested that V. anguillarum may produce multiple AHL signal molecules. Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V. anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis. The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases. Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators. Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed. However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E. coli and C. violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity.