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 μmol/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 μmol/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(Pdx1) 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 μmol/L ATRA+DMEM/F12+10% FBS+1% double antibody under matrigel culture method in the present study.