To explore the altered expressions of embryonic stem-related genes Oct4 and Nanog in pancreatic cancer stem cells (CSCs).The uni-cell suspension of human pancreatic cancer cell line PANC-1 was prepared and incubated with CD24 and CD44 antibodies. Flow cytometer was used to separate CD24(+)CD44(+) pancreatic cancer stem cells. Tumor cell spheres were observed under light microscope. Then CSCs were induced to differentiate with 10% fetal bovine serum and the expressions of CD24 and CD44 re-evaluated by flow cytometer. Finally the cells were divided into 2 groups, group 1: CD24(+)CD44(+) and group 2: non-separated group. RT-PCR (reverse transcription-polymerase chain reaction) and Q-PCR (quantitative-polymerase chain reaction) were used to examine the transcriptions of Oct4 and Nanog in CSCs. The immunofluorescence was employed to examine the expressions of Oct4 and Nanog. Chemo-sensitivity to gemcitabine was determined by CCK8 assay in each group.About 1%-3% CD24(+)CD44(+) CSCs were separated from cell line PANC-1. The sorted cells were cultured in a stem cell culture medium to observe the spheroid-forming capacity. And they showed a higher colony-forming efficiency than the unsorted cells [(122 ± 6)‰, P < 0.05]. When cultured in medium with serum, these cells gradually returned to the status of parental cells with a low expression of CD24 and CD44. Both Oct4 and Nanog were highly expressed in CD24(+)CD44(+) stem cells. And the CD24(+)CD44(+) subgroup demonstrated a higher resistance to gemcitabine.Subpopulation cells CD44(+)CD24(+) have the properties of tumor stem cells. The up-regulated levels of Oct4 and Nanog may be highly correlated with the multi-potency and a higher drug-resistance of pancreatic CSCs.