is the main Vibrio species isolated from diseased mariculture animals with clinical symptoms of bacterial septicemia and skin ulcer in the South China Sea. This bacterium belongs to the family of Vibrionaceae and is normal inhabitant in estuarine and marine environments. Proteome analysis by two dimensional electrophoresistogether with mass finger print is a powerful approach for protein resolution and identification of complex biological samples. In this studyV. alginolyticus in the pI range from 4.0 to 7.0. The techniques for proteome analysis were used to establish reference map ofstrain ZJ03 was cultured in TSB medium for 24 h at 28 . Total soluble proteins were extracted with lysis buffer and purified with a 2-D clean-up kit. Then the purified proteins were labeled with Cy3 or Cy5 and separated by 2-DE. 2-DE gels were scanned with Typhoon 9410 and analyzed by DeCyder 6.0. 2-DE image analysis revealed 902 with matching rate of 96%. The results were obtained from three gels run with 24 cm immobilized pH gradient strips and 12.5% SDS-PAGE gels. Sixty-eight high-abundant spots were chosen for mass spectrometry identification. Among the 68 spots from the 2-DE map，and the other 8 spots matched with the proteins in NCBInr Database. Among the 68 proteinsThe second is the transport and binding proteinsThe third is the outer membrane proteins，purine nucleoside phosphorylase and alkyl hydroperoxide reductase subunit C-like protein were proposed. Two operons are proposed which involved in energy metabolism. Ten pathogenicity associated proteins were found. They are OMP TolCpeptide ABC transporteralanine dehydrogenasealkaline phosphatase. The primary proteome reference map established in this study is helpful in future comparative proteomic investigations on bacterium growth under various experimental conditions or on different bacterial strains.