Objective: To explore the protocol that induced marrow mesenchymal stem cells (MSCS) differentiate into islet-secreting cells in vitro. Methods: Using a defined culture medium and technique for transdifferentiation, MSCs from adult SD rats were guided into specific insulin-secreting cells. The expressions of islet-specific hormones and proteins, such as insulin, glucagon, somatostatin and pancreatic duodenal homeobox I (PDX-1), were analyzed by indirect immunofluorescence cytochemistry staining before and after induction. And the expressions of pancreatic islet cell differentiation-related transcripts, such as nestin, insulin 1, glucose transporter2 (GLUT2), Isl-1, Pdx-1, Pax-4, Pax-6, were detected by reverse transcription-PCR (RT-PCR). In addition, insulin secretion was examined using radioimmunoassay. Results: After induction for 5h, 44.6% +/- 7.3% differentiated MSCs expressed nestin, a marker of neural stem cells, increased 61.8 +/- 8.4% after 24 h, but disappeared at the day of 14. Meantime, islet-like cellular clusters expressed insulin, glucagon, somatostatin and pancreatic duodened homeobox 1 after 14 d and became more apparent by the day of 28. Differentiated cells were found to be immunoreactive to insulin, glucagon, somatostatin and PDX-1, and expressed insulin 1, GLUT2, GK, Isl-1 Pdx-1, Pax-4, Pax-6 mRNA. In addition, the results of cumulative quantities of insulin with 24 h and the stimulation index showed that differentiated cells were able to produce insulin at higher level, and displayed that glucose-dependent insulin released in vitro. Conclusion: Adult rat MSCs can be differentiated to insulin-secreting cells in vitro. This approach might lead to widespread cell replacement therapy for type 1 diabetes.