Background/Aims: The polycomb protein Bmi-1 plays oncogenic roles in various cancers. Here we aimed to investigate the contribution of Bmi-1 on the malignant behaviors of pancreatic cancer such as chemoresistance, invasion and tumorigenesis. Methods and Results: The MTT cell proliferation assay showed that shRNA mediated Bmi-1 knockdown and enhanced the chemosensitivity of pancreatic cancer cells to gemcitabine. The transwell invasion assay showed that Bmi-1 knockdown inhibited the invasion of pancreatic cancer cells in vitro. Notably, the reduced abilities of chemoresistance and invasion were associated with the transition from the mesenchymal phenotype to the epithelial phenotype of pancreatic cancer cells. Moreover, Bmi-1 knockdown led to the inhibition of the PI3K-Akt pathway and disrupted the sphere-forming abilities of pancreatic cancer cells. A nude mouse xenograft experiment demonstrated that pancreatic cancer cells depleted of Bmi-1 showed weak tumorigenicity in vivo. Conclusion: Our data suggest that Bmi-1 plays an important role in the progression of pancreatic cancer and represents a novel target for antitumor therapy of pancreatic cancer.