Objective:To explore epithelial-mesenchymal transition (EMT) in gemcitabine-resistant cancer cell line SW1990/GZ. Methods: Morphological characteristics of SW1990 cells and SW1990/GZ cells were observed by inverted microscope. Cell cycle distribution was analyzed by flow cytometry. The expressions of EMT-associated markers were determined by Western blot. Invasion and migration of cells were detected by transwell. The proportion of cancer stem cells was analyzed by flow cytometry after labeled the surface specific antigen. Results: SW1990/GZ cells exhibited mesenchymal morphology,and no significant G1 accumulation phenotype in SW1990/GZ cells compared with SW1990 cells. SW1990/GZ cells reduced expression of the epithelial marker E-cadherin but increased expression of the mesenchymal marker vimentin. SW1990/GZ cells have greater invasion and migration activity(P < 0.01). Compared with SW1990 cells,the number of CD44+CD24+ cells and CD133+ cells increased by 2 times and 4 times in SW1990/GZ cells(P < 0.01). Conclusion:SW1990/GZ cells have undergone an EMT,and are enriched with cancer stem cells.