Objective To establish a modified induction method for differentiation of rat pancreatic ductal stem cells (rPDSCs) to form islet-like cells. Methods All-trans retinic acid(ATRA) was added at 2, 4, 6 and 8 jjimol/L in the basal culture medium DMEM/F12 + 10% FBS + 1% penicillin/1% streptomycin to induce the differentiation of rPDSCs to form islet-like cells in vitro, and the optimal induction concentration of ATRA was screened. Based on the optimal ATRA induction concentration, rPDSCs were then induced to form islet-like cells in vitro by matrigel culture, suspension culture or hanging drop culture, respectively, to screen the optimal induction culture method . Cell morphology, dithizone(DTZ) staining, cell immunofluorescence staining, Real-time PCR and ELISA were used to detect the induced islet-like cells. Results Compared with the control group, 6 (jumol/L ATRA and matrigel culture were the best in the basic culture medium. After 28 days of induction, the cells enriched and differentiated to form islet-like spherical cell clusters; DTZ staining was positive; Pancreatic duodenal homeobox-1 (Pdxl) and insulin were expressed at gene and protein levels, respectively; Glucose stimulation, release insulin and C-peptide, showed glucose concentration dependent. Conclusion The in vitro differentiation of rPDSCs to form islet-like cells could be successfully induced by using 6 |xmol/L ATRA + DMEM/F12+10% FBS+1% double antibody under matrigel culture method in the present study.