Abstract: Spotted longbarbel catfish Mystus guttatus is a precious species of fish in Pearl River. However,gill-rotted dis-ease caused very high mortality in cultured spotted longbarbel catfish in recent years and brought huge economic losses. To protect the fish, many researches have been carried out on pathogenic bacteria of the gill-rotted disease. In this study,a vir-ulent strain of bacteria Mg2 was isolated from the rotted gill of diseased spotted longbarbel catfish. Artificial infection test showed that Mg2 could lead to 100% mortality of fish in the experimental group with the same signs as the natural infected fish and was the causative pathogen associated with the disease. The bacterium grew very well in 0.5% peptone liquid me-dium at 25-28℃ under pH 6.5-7.5,and then there was no growth at 4℃ or more than 0.6% of NaCl content. The bac-terial colony has yellow,irregular shape with undulated margin in 0.5% peptone solid medium. Electron microscope obser-vation revealed that Mg2 was in the absence of flagella and capsule,gram negative, and exhibits rod-shaped morphology with a size of 0.5×6.5-11μm. Physiological and biochemical analysis showed that it had the characteristics of the oxi-dase, catalase and nitrate reduction positive; hydrolyzation of casein and gelatin; non-hydrolyzation of cellulos,chitin, tyro-sine,aesculin and starch;non-fermentation of indole and glucose. Drugs sensitivity tests showed that it was highly sensitive to baytril,norfloxacin,nalidixic acid, erythromycin, lincomycinum. Polymerase chain reaction (PCR) of 16S rRNA gene was carried out by using the primers specific for the Mg2 strain and sequences alignments displayed that Mg2 had the high-est homology in the 16S rRNA nucleotide level with Flavobacterium columnare strain Ga-6-93, Flavobacterium columnare strain and Flavobacterium columnare strain LV339-01 with the values 99.6%,99.8% and 99.7%,respectively,while had 97.5% homology with M165. Phylogenetic analysis was constructed based on the 16SrRNA gene of Flavobacterium colum-nare in the GenBank database using Vector NTI9.0 software and revealed that the strain Mg2 clustered with Flavobacteri-urn columnare strain Ga-6-93. Therefore,it indicated that the Mg2 strain should belong to the members of Flavobacterium columnare according to the morphological features, physiological and biochemical analysis and 16S rRNA gene homology comparison of the bacteria. To avoid gill-rotted disease, moderate density and regular disinfection in fish farm is the practi-cal method of controlling the disease