Outbreaks of mass mortality among cultured juvenile Chinese shrimp Fenneropenaeus chinensis occurred during summer 2010 in Jiangsu Province, PR China. Gross signs of disease included lethargy, swimming near the water surface and close to pond edges, pale red shell and appendages, breaking of antenna, and reduction in food consumption. Green and yellow bacterial colonies were recovered on thiosulfate citrate bile salt sucrose agar plates from the diseased shrimp. The predominant observed colonies were purified (VP1 and VR1) and their virulence toward postlarvae of Fenneropenaeus chinensis confirmed by bath experimental challenges. The phenotypic characteristics of the isolates, including morphological, physiological, and biochemical traits, were determined. Three housekeeping genes (16S rRNA, RNA polymerase a-chain (rpoA), and DNA gyrase B subunit (gyrB)) were sequenced, and phylogenetic trees based on rpoA and gyrB sequences were constructed. Phenotypic and genetic results confirmed the diseased shrimp were infected with both Vibrio parahaemolyticus and Vibrio rotiferianus. To our knowledge, this is the first report of V. rotiferianus as a pathogenic bacteria in shrimp. In addition, the activities of extracellular enzymes and hemolysin were also studied. Results showed that all the isolates (VP1–VP3 and VR1–VR3) produced β-hemolysis on rabbit blood agar, lecithinase, proteinase, and gelatinase, but DNase and lipase were not produced.