Edwardsiella tarda is an intractable Gram-negative pathogen in many fish species to cause edwardsiellosis. Its infection leads to extensive losses in a diverse array of commercially important fish. The type III secretion system (T3SS) has been considered as one of the major virulence factors and plays important roles in its intracellular lifestyle. In this study, an E. tarda EIB202 mutant WED with deletions in the T3SS genes for EseB, EseC, EseD and EscA, along with the aroC gene for the biosynthesis of chorismic acid, as well as the curing of endogenous plasmid pEIB202 was constructed by allelic exchange strategy. Compared to the wild-type EIB202 which was highly virulent towards turbot (Scophthamus maximus) via intraperitoneal (i.p.), intramuscular (i.m.) injection or immersion and caused systemic infection in turbot as well as the unexpected red mouth symptom when immersion challenged, WED was highly attenuated when inoculated into turbot via i.m., i.p. and immersion routes, and exhibited significantly impaired capacity to survive in fish tissues. WED showed 5700-fold higher 50% lethal dose (LD50) than that of the wild type when i.m. or i.p. challenged. Inoculation with WED by i.p. or immersion injection routes elicited significant protection against the challenge of the wild-type E. tarda after 5 weeks of vaccination. The vaccinated fish produced low while significant level of specific antibody and showed increased expression of immune-related factors including IL-1β, IFN-γ, MHC II, MHC-I and CD8, indicating that WED possesses significant immunoprotective potential. Furthermore, our data indicated that a single dose of i.p. and immersion vaccination with WED could produce significant protection as long as 12 and 6 months, respectively. These results demonstrated the feasibility of WED as a live attenuated vaccine in turbot against edwardsiellosis by immersion or i.p. injection routes.