Abstract: The grass carp (Ctenopharyngodon idellus) is very important in aquaculture in China. However, the grass carp is often infected with bacterial diseases, resulting in huge economic losses in recent years. This paper reported an acute haemorrhagic disease of Ctenopharyngodon idellus in Henan Province, China. The typical symptoms of the disease included decay of dorsal and caudal fins, congestion and hemorrhage in skin, muscle and intestine and ascites. The aim of this research was to isolate the pathogens of bacterial haemorrhagic disease in grass carp and to determine their LD50 in grass carp, and also to identify the sensitivity to antibiotics. The pathogen were isolated from gill, liver, spleen, kidney and ascites of infected grass carps, respectively, and purified in Shieh medium and LB medium. The shape of bacteria was observed by either optical microscope or electronic microscopic. And the physiological and biochemical identification of the bacteria was performed on VITEK 2 automated microbiology system (Systems version: 0301, Mrieux). Besides, the sequences of 16S rRNA were amplified by PCR with universal primers and the sequences were analyzed by BLAST. Correspondingly, the phylogenetic trees were constructed by MEGA 4 based on the 16S rDNA sequences. To confirm the pathogenesis of the isolated strains, the artifial infection and half of lethal dose test were conducted in healthy grass carps. The result showed that three kinds of bacteria were isolated from the diseased grass carp with typical syndrome, and were named as F2, F4 and XF, respectively. The colonies of F2 and F4 were round, white and moist, while the colony of XF was rhizoid-like, yellow and dry. The result of optical microscopy showed that all the three strains were arc-rod and Gram-negative. There were flagellums on the polar of the F2 and F4 based on the result of electronic microscopy. The F2, F4 and XF pathogens were identified as Vibrio cholerae, Aeromonas hydrophila and Flavobacterium columnare, respectively, which based on the result of morphological observation, biochemical test, with their 16S rDNA sequences submitted into GenBank as the following accession numbers, GU296110 (F2), GU296111 (F4) and GU296112 (XF). The phylogenetic trees based on 16S rDNA sequences showed that the F2 was clustered closely with Vibrio cholerae strain O395, and XF with Flavobacterium columnare IAM14301. The typical symptoms of the disease such as decay of dorsal and caudal fins, congestion and hemorrhage in skin, muscle and intestine with ascites appeared in artificially infected grass carp, with 100% mortality observed. All the three strains were proved to be the pathogens of bacterial haemorrhagic disease in grass carp. The LD50 of V. cholerae and A. hydrophila to healthy grass carp by intraperitoneal injection challenge was 0.15104 cfu/g and 0.96103 cfu/g, respectively. The antibiotic sensitivity analysis showed that V. cholera was sensitive to rifampicin, levofloxacin, streptomycin, ofloxacin and gentamycin, A. hydrophila to ceftriaxone, ofloxacin and gentamycin, and F. columnare to ampicillin, rifampicin, levofloxacin, azithromycin, streptomycin, tetracycline, erythromycin, benzylpenicillin, ofloxacin and ciprofloxacin. However, the culture of these three strains was only inhibited by ofloxacin.