Edwardsiella tarda is a Gram-negative enteric bacterium affecting both animals and humans. Recently, a type III secretion system (TTSS) was found in Ed. tarda. Such systems are generally used by bacterial pathogens to deliver virulence factors into host cells to subvert normal cell functions. Genome-walking was performed from the eseB and esrB genes (homologues of Salmonella sseB and ssrB,respectively) identified inprevious studies, to determinethe sequences of the TTSS. Thirty-five ORFs were identified which encode the TTSS apparatus, chaperones, effectors and regulators. Mutants affected in genes representing each category were generated and found to have decreased survival and growth in fish phagocytes. LD50 values of the mutants wereincreasedbyatleast10-foldincomparisontothoseofthewild-typestrain.Theadherenceand invasion rates of the esrA and esrB mutants were enhanced while those of the other mutants remained similar to the wild-type. The eseC and eseD mutants showed slight autoaggregation in Dulbecco’s Modified Eagle Medium, whereas the rest of the mutants failed to autoaggregate. RegulationoftheTTSSwasfoundtoinvolvethetwo-componentregulatorysystemesrA–esrB.This study showed that the TTSS is important for Ed. tarda pathogenesis. An understanding of this system will provide greater insight into the virulence mechanisms of this bacterial pathogen.