Aeromonas infections are the most common bacterial disease of cultured fish in China. In this study, a gram-negative bacillus was isolated from the liver of diseased koi carp (Cyprinus carpio koi), and named strain KC-2. The results of morphological and biochemical tests, as well as phylogenetic analysis derived from 16S rRNA and gyrB sequences indicated that the isolated strain KC-2 was highly identical to the known Aeromonas media ATCC 33907. Experimental infection assays were conducted, and pathogenicity was demonstrated in crucian carp (Carassius auratus) and zebrafish (Danio rerio). Antimicrobial susceptibility testing showed that the strain KC-2 was sensitive to cefalotin, cefixime, cefotaxime, gentamicin, netilmicin, azithromycin and chloramphenicol. This is the first report on the isolation and identification of A. media from diseased, cultured koi fish. The results of the study will provide a scientific reference for prevention of bacterial disease of koi carp and identification of A. media in fish.