Edwardsiella ictaluri is a Gram-negative bacterium and the causative agent of enteric septicemia of catfish. In this study, we examined the expression and function of the LuxS from a pathogenic E. ictaluri strain, 1901. J901 was found to produce autoinducer 2 (AI-2) activity that maximized at mid-logarithmic phase and was enhanced by glucose and repressed by high temperature. Consistently, a luxS gene (luxSEi) was identified in J901, whose expression was regulated by cell density, glucose, and temperature in a manner similar to that observed with AI-2 activity. Further analysis showed that LuxSEi is a biologically active AI-2 synthase that was able to complement the luxS-defective phenotype of Escherichia coli DH5alpha. To examine the functional importance of LuxSEi, a genetically modified variant of J901, J901Ri, was constructed, in which luxSEi, expression was blocked by RNA interference. Compared to the wild type, J901Ri was (i) reduced in AI-2 activity to a level of 59% of that of the wild type; (ii) impaired in both planktonic and biofilm growth; (iii) significantly attenuated in the ability to infect cultured fish cells and to cause mortality in infected fish; (iv) unable to induce the expression of certain virulence-associated genes. Addition of exogenous AI-2 failed to rescue the growth defect of J901Ri as free-living cells but restored biofilm production and the expression of virulence genes to levels comparable to those of the wild type. Taken together, these results indicate that LuxSEi is a functional AI-2 synthase that is required for optimal cellular growth and host infection.