In a previous study, an HBsAg gene-bearing transgenic tomato line was made available and it exhibited notable physiological alterations compared with the non-transgenic tomato (control). In particular, leaves of the transgenic plants were fleshy and dark. We hypothesized that a change in leaf proteins of the transgenic plants account for the observed phenotypes. In this study, total protein content in leaves of the transgenic plants was analyzed by 2-dimensional difference gel electrophoresis. A total number of 700 protein spots were detected on silver-stained gels, of which 368 protein spots were matched between the control and sample gels. Among these matched proteins, the expression levels of 122 proteins in the transgenic plants were upregulated while those of the rest were downregulated. In addition, 25 abundant proteins (value ratio > 2.0) on silver-stained gels were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Sixteen differentially expressed proteins were identified, of which 13 were predicted to be involved in cell division, energy metabolism, protein synthesis and processing. The possible roles of these proteins in the transgenic tomato strain have been discussed. Taken together, our data indicate that significant alterations in protein expression occur in transgenic tomatoes bearing the HBsAg gene. Our findings will help broaden our knowledge of the mechanism by which exogenously expressed genes lead to phenotypic alterations in transgenic plants.