Objective To investigate the effects of Oct4 and Nanog gene silenced by shRNA on drug resistance of pancreatic cancer stem cells and explore new ways of improving chemotherapeutic effect on the pancreatic cancer. Methods The CD44+CD24+ESA+ pancreatic cancer stem cells (PCSCs) were isolated from PANC-1 cell line by flow cytometry and constructed cell strains in vitro. We constructed lentiviral virus-shRNA-Oct4/Nanog vector target Oct4 and Nanog and silenced them simultaneously, and then identified whether PCSCs inhabited expressions of the two genes (PCSCs-shOct4+shNanog) showed the changes of drug resistance to gemcitabine by cell counting Kit-8 (CCK-8) cell proliferation assay and the expression of ABCG2 were examined by Real-Time RT-PCR and Western blotting analysis. Results The high expressions of Oct4 and Nanog were in CD44+CD24+ESA+ PCSCs, which indicated enhanced resistance to gemcitabine in vitro. Double knockdown of Oct4 and Nanog significantly reduced stemness of PCSCs, expression of ABCG2, and chemoresistance to gemcitabine. The differences were statistically significant (P<0.05). Conclusion The results suggest that Oct4 and Nanog play critical roles in maintaining the stemness of PCSCs. Gene silencing of Oct4 and Nanog may inhabit the drug resistance of PCSCs.