The epithelial-mesenchymal transition plays a crucial role in the progression of pancreatic cancer. The aim of this study was to examine the possible association between the epithelial-mesenchymal transition and cancer stem-like cells in pancreatic cancer. We used transforming growth factor β to induce an epithelial-mesenchymal transition. The proportion of pancreatic cancer stem-like cells was measured and sorted by flow cytometry. The expression of markers was measured by quantitative PCR and Western blot analysis. Cell cycle distribution was assessed by flow cytometry. We evaluated the migration and invasion activity by Transwell tests. The proportion of pancreatic cancer stem-like cells was significantly increased following transforming growth factor β treatment. Cells were sorted in culture, the cancer stem-like cells exhibited a higher degree of epithelial-mesenchymal transition and demonstrated upregulation of vimentin, a mesenchymal phenotypic marker, compared to the CD44(-)CD24(-) cells. Pancreatic cancer stem-like cells exhibited greater invasion and migration activity in vitro compared to the CD44(-)CD24(-) cells. These results suggested a direct link between epithelial-mesenchymal transition and cancer stem-like cells in pancreatic cancer.