Bone marrow mesenchymal stem cells (BM-MSCs) have the capacity for renewal and the potential to differentiate in culture into several cell types including osteoblasts, chondrocytes, adipocytes, astrocytes, myocytes, oligodendrocytes, and neurons. Albeit previous reports demonstrated some of the effects of extensive subculturing on MSCs, the results still remain controversial. The aim of this study was to generate proteome maps of undifferentiated rat BM-MSCs, and identify differentially regulated proteins during serial subcultures within the first 10 passages. Proteins extracted from Wistar rat BM-MSCs were separated by two-dimensional gel electrophoresis and about 1000 protein spots were detected using the Sypro Ruby dye. Among them, 106 selected spots were digested with trypsin for mass spectrometry analysis, and 31 proteins were successfully identified by MALDI-TOF-MS. Here, 18 differentially expressed proteins are reported for the first time; these proteins are classified into 8 functional categories: metabolism, signal transduction, cell adhesion and growth, cytoskeleton, cell cycle, protein degradation, cell-cell interaction, and ion transfer. These proteins are reported to be involved in cell proliferation and differentiation through different signaling pathways. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of proliferation potential under serial subculturing. The molecular mechanisms of rat BM-MSCs are discussed at the proteome level.