The outbreak of mass mortality of Japanese flounder occurred in an aquaculture farm in Hebei province of China. This study isolated and identified Pseudomonas putida as the dominant bacterium from diseased Japanese flounder (Paralichthys olivaceus) based on morphological, physiological, biochemical characteristics, 16S rRNA gene sequencing, and whole-genome sequencing. Pathogenicity assessment, histopathological analysis, and host immune response were investigated. Results demonstrated that P. putida was pathogenic, causing acute enteritis and multiple organ damage in infected fish. The median lethal dose (LD