Pancreatic cancer stem cells play a crucial role in tumorigenesis and chemoresistance. The Hedgehog signaling pathway is a key regulator in pancreatic tumorigenesis and drug resistance. To identify pancreatic cancer stem cells, tumorspheres derived from the PANC-1 pancreatic cancer cell line were cultured under a floating-culture system. PANC-1 tumorspheres possessed properties of self-renewal, differentiation, higher tumorigenesis and chemoresistance. It was observed that Hedgehog pathway is active in PANC-1 tumorspheres as shown by expression of hedgehog components Smo, Gil 1 and Gli 2, detected by quantitative RT-PCR and western blotting. After cyclopamine-mediated blockade of hedgehog, a decrease in proliferation of PANC-1 tumorspheres and G0/G1 transition were observed, as well as a decreased expression of Bmi-1 in PANC-1 tumorspheres. Cyclopamine reversed chemoresistance to gemcitabine, resulting in decreased expression of ABCG2 in PANC-1 tumorspheres. Taken together, our data indicate that PANC-1 tumorspheres have 'stemness' potential, and hedgehog signaling pathway plays an important role in the regulation of self-renewal and reversal of chemoresistance in cancer stem cells in pancreatic adenocarcinoma.