Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 1.0×105 colony forming units (CFU)mL-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillarum with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay (IFA) was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.