Pseudomonas plecoglossicida has been reported as the causative agent of the visceral granulomas disease in farmed Larimichthys crocea. The pathogenic strain NB2011 encodes a typical type Ⅲ secretion system, which is present in many Gram-negative pathogens, and may be an important virulence factor of this strain. ExsA, the transcription regulator of this system, is essential for the type Ⅲ expression and function. In this study, the mutant strain ΔExsA NB2011 was obtained by the method of double homologous recombination whose internal sequence of the ExsA gene of NB2011 was replaced by a kanamycin resisting gene. Adhesion, internalization of macrophage J774, and intracellular replication of the mutant strain were detected, LD₅₀ of artificial challenge was tested, and the ultrastructure pathology of infected fish was investigated. Compared to the wild strain, the number of the internalized mutant strain in the macrophages was a little lower, the number decreased in several hours and was too low to be detected in 12 h; while the number of wild strain increased after 12 h post internalization; LD₅₀ of the mutant strain increased to over 190 times the wild strain; TEM observation results showed the wild bacteria survived and replicated in macrophages of the infected fish, while no mutant bacteria were observed in the fish tissue during the experiment time. The results indicated a dramatic virulence attenuation of ΔExsA NB2011. The strain may serve as a good candidate for vaccine development.