ABSTRACT: The Type III secretion system (T3SS) is essential for intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals. Two possible effector candidate genes (eseE and eseG) and 7 hypothetical genes (esaB, escC, orf13, orf19, orf26, orf29, and orf30) located in the T3SS gene cluster were inactivated by an allelic exchange method, and we found that E. tarda strains carrying insertion mutations in escC, orf13, orf19, orf29, and orf30 were unable to replicate within J774 macrophages and HEp-2 epithelial cells. Furthermore, the virulence of these mutants to zebrafish was severely attenuated as well. These data suggest that the escC, orf13, orf19, orf29, and orf30 genes are required for intracellular replication and virulence of E. tarda.