Aims: The aim of this study was to generate temperature-sensitive Edwardsiella tarda mutant and to evaluate potential of the mutant as a vaccine in olive flounder (Paralichthys olivaceus). Methods and Results: A temperature-sensitive E.similar to tarda mutant was generated by replacement of alr gene promoter with cI857-?PR promoter system plus another CI857 expression cassette that was driven by a constitutive promoter of E.similar to tarda (EtPR C28-1). Growth of the mutant strain was not different to that of wild-type E.similar to tarda under conditions of culture at 39 degrees C. However, growth of the mutant strain was retarded by culturing at 25 or 20 degrees C without d-alanine. To further inhibit leakage of ?PR promoter, the mutant strain was transformed with a vector harbouring an EtPR C28-1-driven cI857 cassette (pEtPR-cI857), which resulted in more limited growth compared to the mutant without the plasmids. The level of alr gene transcription in the mutant E.similar to tarda and the mutant harbouring pEtPR-cI857 was well coincided with the result of bacterial growth. Olive flounder fingerlings immunized with the E.similar to tarda mutant showed decreased mortality, and a boost immunization induced 100% protection against E.similar to tarda infection. The protection rate of fish was proportional to the serum agglutination titre against E.similar to tarda. Conclusions: An attenuated E.similar to tarda mutant induced by shifting down temperature below 30 degrees C was firstly generated. Immunization of fish with the mutant led to protection against virulent E.similar to tarda challenge. Significance and Impact of the Study: The results suggest that the present temperature-sensitive E.similar to tarda mutant can be a good candidate for effective vaccines for prophylaxis of edwardsiellosis. Moreover, as the present E.similar to tarda mutant can be cultured without supplementation of the specific nutrient and can be attenuated just by decreasing temperatures below 30 degrees C, vaccines based on the present E.similar to tarda mutant would be advantageous in an economical aspect.