Cancer stem cells (CSCs) comprise a small population of cells in cancerous tumors and play a critical role in tumor resistance to chemotherapy. miRNAs have been reported to enhance the sensitivity of pancreatic cancer to chemotherapy. However, the underlying molecular mechanism requires better understanding. Cell viability and proliferation were examined with CCK8 assays. Quantitative real-time polymerase chain reaction was executed to assess mRNA expression. StarBase database was used to select the target genes of miRNA, which were further affirmed by dual luciferase assay. Transwell assay was used to analyze cell invasion and migration. We proved that miR-497 could be obviously downregulated in pancreatic cancer tissues and CSCs from Aspc-1 and Bxpc-3 cells. In addition, inhibition of miR-497 evidently accelerated pancreatic CSC gemcitabine resistance, migration and invasion. Moreover, we revealed that nuclear factor kappa B 1 (NFκB1) was prominently upregulated in pancreatic cancer tissues and pancreatic CSCs, and NFκB1 was also identified as a direct target of miR-497. Furthermore, we demonstrated that overexpression of NFκB1 could also notably promote the viability, migration, and invasion of gemcitabine-treated pancreatic CSCs, but this effect could be partially abolished by miR-497 overexpression. Those findings suggest that miR-497 overexpression could suppress gemcitabine resistance and the metastasis of pancreatic CSCs and non-CSCs by directly targeting NFκB1.