In May 2017, there was a massive mortality of mud crab Scylla paramamosain in a farm of Sanmen, Zhejiang Province, China. The bacterial isolates from the hepatopancreas and muscle of the diseased S. paramamosain showed the same phenotypic traits and biochemical profiles, and were identified as Photobacterium damselae subsp. damselae in accordance with the biochemical characteristics and molecular identification with the 16S rRNA and ureC genes. The morphological observation suggested that P. damselae subsp. damselae QX175062 strain was a rod-shaped bacterium with singlet flagellum. Bacterial infection tests were conducted by intramuscular injection of the QX175062 isolate into healthy mud crabs, and the results indicated that the isolate was lethal to S. paramamosain with the LD50 value of 3.63 × 101 CFU/g (body weight). The challenged mud crabs showed similar pathological signs as those that were naturally infected. Histopathological analysis of mud crabs injected with QX175062 isolate showed agglutination and necrosis of hepatopancreatic tubules in hepatopancreas; ruptured or disordered tissue and fiber dissolution in muscle. Antibiogram study revealed that QX175062 isolate was mainly sensitive to tetracyclines, aminoglycosides, macrolides and quinolones, but resistant to β-lactam antibiotic drugs. This first case report underlines the involvement of P. damselae subsp. damselae in the mud crab aquaculture with disease outbreak and emphasizes the need of developing rapid diagnostic methods and prevention measures to reduce the economic loss caused by P. damselae subsp. damselae infection in mud crab aquaculture.