In this study, we first sought to determine the existence of side population (SP) cells in pancreatic cancer cell lines. Furthermore, we compared the biological characteristics of SP and non-SP cells. The presence of side population cells in pancreatic cancer cell lines was detected by Hoechst 33342 staining and FACS analysis. Cell cycle distribution was analyzed using flow cytometry. SP and non-SP cells were exposed to various concentrations of gemcitabine; drug sensitivity was examined using the MTT assay and flow cytometry using Annexin-V and PI staining. To compare the tumorigenic ability in vivo, groups of nude mice were orthotopically inoculated with varying numbers of SP and non-SP cells. The percentages of CD44+CD24+ and CD133+ in SP and non-SP cells were also detected by FACS analysis. The SP fraction was detected in BxPc-3, CFPAC-1, MIA PaCa-2, PANC-1 and SW1990 pancreatic cancer cell lines. Cell cycle analysis revealed that the SP cells contained more cells in the G1 phase and fewer cells in the S phase when compared with the non-SP cells. The SP cells exhibited increased tumorigenetic ability following in vivo transplantation into BALB/C nude mice and increased chemoresistance following in vitro exposure to gemcitabine. FACS analysis showed that the SP cells contained more CD44+CD24+ and CD133+ cells than the non-SP cells. In conclusion, these observations suggest that SP cells in the pancreatic cancer cell lines possess the property of cancer stem cells. SP cells may therefore be novel specific targets for the effective treatment of pancreatic cancer.