Abstract: To investigate the resistance mechanism of different tobacco cultivars to Ralstonia solanacearum, the whole proteins of seeding leaves of high resistance cultivars DB101 and susceptible cultivars Honghuadajinyuan were compared using two-dimensional electrphoresis. A total of 26 proteins were found to be differentially expressed between the two cultivars. Among them 12 protein spots were up-regulated in DB101 and 14 proteins were up-regulated in Honghuadajinyuan. Next, 22 differentially proteins were identified by mass spectrometry. Base on the metabolic pathway and biochemistry functional, we grouped them into 6 clusters: photosynthesis, metabolism and energy, redox homestasis, expressional regulation, defensive responses proteins and putative protein. Then a complex molecular mechanism and metabolic regulative network in the resistant reponses to the infection of Ralstonia solanacearum has been outlined in this research. In these differentially expressed proteins/enzymes, glutamate-1-semialdehyde 2,1-aminomutase and diaminopimelate decarboxylase, which were significant upregulated in resistant cultivar DB101, have been identified for the first time besides the pathogenicity proteins reported previously. To our knowledge, this was the first attempt to explore the resistance mechanism of tobacco to the Ralstonia solanacearum on a proteome level, and differently-expressed proteins might play an important role in further research of tobacco resistant breeding and functional cloning of the resistant-related genes in tobacco.