Rodents respond to a chronic high-fat diet (HFD) in two ways: some readily become obese (obesity prone, OP) and others do not (obesity resistant, OR). Although several hypotheses have been proposed, the mechanisms underlying the inter-individual susceptibility to diet-induced obesity remain to be fully defined. In the present study, two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption ionisation time-of-flight MS was carried out for identification of differentially expressed liver proteins in OP and OR rats fed a HFD, in an attempt to discover marker proteins involved in susceptibility and/or resistance to obesity in rat liver. The 2-DE analysis demonstrated that forty spots from 380 visualised spots were differentially regulated between the groups. Among these forty spots, twelve were differentially expressed proteins between OP and OR rats, reaching statistical significance. Of these, five proteins have already been linked to obesity; however, seven proteins involved in obesity susceptibility or resistance were identified for the first time in the present study. In order to validate the proteomic results and gain insight into the metabolic changes between the OP and OR groups, we further confirmed the expression pattern of some proteins of interest by Western blot analysis. Combined results of proteomic analysis with Western blot analysis revealed that reduced lipogenesis and increased fat oxidation were achieved in the livers of OR rats. In conclusion, the present proteomic study is an important advance over the previous steps required for identification of OP and OR rats, and should prove valuable in the search for the pathogenesis of obesity in humans.