Objective To identify pancreatic duct-derived stem cells （PDSCs） from the pancreatic duct of rats, and culture in three-dimension cell culture system or two-dimension cell culture, with characterization on their morphology and phenotype. Methods Adult male Wistar rats underwent perfusion with collagenaseⅤvia the pancreatic duct, the pancreas was surgically procured, digested, followed by discontinued density gradient centrifuge to isolate islets from acinar and ductal tissue. The acinar and ductal tissues were cultivated in serum-containing medium in the three-dimension or two-dimension cell culture seperatedly to obtain adherent cells, as PDSCs, which were expanded by consecutive passages. The cell junction, cell cycle, and cell apoptosis rate between the two-dimension and three-dimision cell culture were compared by means of microscope and flow cytometry. Results PDSCs of rats showed direct adherence culture in three-dimension and two-dimision cell culture systerm. PDSCs grew in many layers in three-dimension cell culture system on 2 days after culture, and possessed capacity of high proliferation on 7 days. Cells were adherent to the bottom of flask on 5 days after culture in two-dimision cell culture. Cell junctions were more obvious in three-dimension cell culture system. Compared with the two-dimision cell culture, the cells of G1 phase increased〔（56.46±1.17） % vs.（37.33±1.21）%〕, and the cells of G2 phase decreased〔（14.62±0.15） % vs. （32.40±1.21）%〕in three-dimension cell culture system （P＜0.01）. The early period apoptosis rate was lower in three-dimension cells culture system than that in two-dimision cell culture 〔（1.48±0.39）% vs. （5.24±1.33）%, P＜0.05〕. The late period apoptosis rate was no statistically significant difference between the three-dimension and two-dimension cell culture （P＞0.05）. Conclusions PDSCs of rats could be obtained in the three-dimension cell culture system. There is cell junction in three-dimension culture mode, which shows a significant enhancement on intercellular interaction, and the biological characters are different between the three-dimension and two-dimension cell culture.