Brucellae are facultative intracellular gram-negative bacteria that cause human disease and significant worldwide economic loss due to infection of livestock. Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in Brucella membrane proteins (MPs). In this study, an immunoproteomic approach was utilized to identify novel antigenic candidate proteins from Chinese Brucella vaccine strain M5 membrane proteins. The membrane proteins were separated by 2-DE and analyzed by western-blotting for their reactivity with the antiserum obtained from goats naturally infected by Brucella. Of a total of 9 immunogenic proteins identified, 7 were shown to be the novel antigens for Brucella spp. Some of the major protein components include outer-membrane protein OMP25, OMP31, and several new immunogenic proteins were identified, such as isovaleryl coenzyme A dehydrogenase, nitroglycerin reductase, succinyl-CoA synthetase subunit and S- adenosine -L- homocysteine hydrolase. Comparing these gene sequences revealed that 8 out of 9 immunoreactive protein genes were found in all 5 different Brucella strains. The elucidation of the immunome of Brucella vaccine strain M5 MPs identified a number of candidate proteins for developing vaccines against Brucella infection in livestock.