Objective: To investigate the expression and action of microRNA-200c (miRNA-200c) in pancreatic cancer stem cells. Methods: Pancreatic cancer stem cells were sorted from human pancreatic cancer PANC-1 cells by FACS using CD24+CD44+ESA+ as marker, and their stem cell properties were assessed by xenograft tumor assay in NOD/SCID mice. The miRNA-200c expression and invasion ability in PANC-1 cells, pancreatic cancer stem cells and pancreatic cancer stem cells transfected with miRNA-200c precursor sequence or negative control sequence were determined by RFQ-PCR method and Transwell invasion assay, respectively. Results: The CD24+CD44+ESA+cells (accounting for 0.8%) sorted from PANC-1 cells presented tumor stem cell properties, and the volume of the xenograft tumor after their subcutaneous transplantation in mice was significantly larger than that after PANC-1 cells transplanted at the same time [(1 725.14±261.29) mm3 vs. (479.65±99.67) mm3, P<0.05]. In pancreatic cancer stem cells compared with PANC-1 cells, the miRNA-200c expression level was significantly decreased (0.15±0.01 vs. 1.00±0.09, P<0.05), and transmembrane cell number was significantly increased (321±7.62 vs. 70±16.47, P<0.05), but the miRNA-200c expression level was significantly increased and transmembrane cell number was significantly decreased in pancreatic cancer stem cells after transfection with miRNA-200c precursor sequence (both P<0.05). Conclusion: MiRNA-200c expression is reduced in pancreatic cancer stem cells, and miRNA-200c has inhibitory effect on growth and invasiveness of pancreatic cancer stem cells.