Objective: To isolate, culture and identify the mouse adult pancreatic ductal stem cells in vitro and to observe the potency of these multipotentional cells differentiation into insulin-producing cells. Materials and Methods: Under anesthesia, mid line laparotomy incision was done then the proximal common bile duct is incised and cannulated with a catheter and injected with enzyme solution. After total pancratectomy, the pancreas is incubated in the enzyme solution. After isolation of pancreatic ductal stem cells followed culture in serum and serum free medium with additional Keratinocyte growth factor. The cells were induced by glucose. Then the cell types of undifferentiated and differentiated cells were identified using immunocytochemical and RT-PCR staining. Results: The pancreatic ductal stem cells cultured in serum free medium grew very slowly. The identification of these cells was then identified by using the pancreatic ductal stem cell marker cytokeratin 19. But in the serum containing media, fibroblast very fastly grown and covered on pancreatic ductal stem cells. The immunoreactive staining and RT-PCR technique showed that pancreatic stem cells stimulated effectively to produce insulin producing cells in vitro. Conclusion: This study revealed the expansion of adult mouse pancreatic ductal tissue in vitro. These cells can be cultured and induced by glucose and differentiated into insulin producing cells.