In order to reveal proteins differentially expressed in the process of rat pancreas development, two-dimensional gel electrophoresis and mass spectrometry were performed on rat pancreas at different stages. After rat pancreas microseperated, protein extracted from of four stages: 15.5 embryonic days (E15.5), E18.5, newborn and adult were separated by immobilized pH gradient two-dimensional gel electrophoresis. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS)and ESI-MS were performed with the proteins which were found to be significantly different presented in these four maps, 8 of which were down-expressed and 1 of which up-expressed at the adult stage, 4 of which specially expressed at adult stage while 6 of which specially expressed in E18.5 stage. All together 18 protein spots were identified. 7 spots were identified as alpha fetoprotein (AFP). 5 spots were pancreatic lipase related protein 1 precursor. 1 spot was tublin beta 5.2 spots were protein disulfide isomerase. The other 3 spots were similar to FLN29 gene product, Trypsin V-A precursor, and Peroxiredoxin 4.Among them, AFP specially expressed in new born and E18.5 stage. The development of pancreas gave rise to the changes of the protein expression patterns. This helps to understand the molecular mechanisms of rat embryonic pancreatic development.