In order to examine potential molecular markers of pancreatic stem cells, a regeneration pancreatic model was induced by 90% partial pancreatectomy (Px) in rats. Changes in the protein expression in rat regeneration pancreas at 3rd day after Px, as comparing to sham surgery (Sx), were analyzed by using two-dimensional gel electrophoresis (2DE), mass spectrometry (MS), and peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. HSP47 and Vimentin were analyzed in Px and Sx rat with Western blotting to confirm the results of 2DE. The average spots in gels for Sx and Px pancreatic tissues were (1 369 ± 31) and (1 315 ± 28), respectively. 2DE displayed 91 spots with at least 1.5-fold of differential expression at the time point of 3 days after pancreatectomy and 53 differentially expressed proteins were identified by PMF. Among them, Vimentin, CK8, L-plastin, hnRNP A2/B1 and AGAT are associated with embryogenesis and cells differentiation and may be new potential pancreatic stem cells markers. The proteome profiling technique provided a broad-based and effective approach for discovering potential biomarkers of pancreatic stem cells.