We used flow cytometry and a DNA-binding dye efflux assay to isolate a side population (SP) of cells with stem cell characteristics from the human pancreatic carcinoma cell line, PANC-1. Non-obese diabetic/severe combined immunodeficiency mouse xenograft experiments showed that SP cells were enriched in tumor initiating capability compared with non-SP cells. Cultured SP cells were able to differentiate into daughter cells and non-SP cells, through asymmetric division. Our study demonstrated that SP cells had high drug-resistance, both in vivo and in vitro. SP cells also showed significantly higher levels of mRNA expression for CD133, ABCG2 and Notch1, when compared to non-SP cells. Furthermore, xenografted tumors derived from injected SP cells and treated with gemcitabine had more CD 133+ cells than untreated ones. We therefore suggest that these SP cells from the PANC-1 cell line were enriched with cancer stem cells.