B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) is a member of the polycomb group of transcriptional repressors. Until now, its expression and functional significance in pancreatic carcinogenesis was unknown. In the present study, we demonstrated that expression of BMI1 was markedly up-regulated in pancreatic cancer cell lines and surgically resected cancer specimens. In addition, BMI1 expression levels correlated positively with the presence of lymph node metastases and negatively with patient survival rates, suggesting a role for BMI1 in the progression of pancreatic cancer. Furthermore, stable down-regulation of BMI1 suppressed cell growth, delayed the G1/S transition, and enhanced the susceptibility of different pancreatic cell lines to apoptosis following expression of a lentiviral-mediated shRNA targeted for BMI1. Expression of the short-hairpin RNA also correlated with the up-regulation of p21 and Bax and the down-regulation of cyclin D1, cyclin-dependent kinase (CDK)-2 and -4, Bcl-2, and phospho-Akt. Finally, growth suppression following BMI1 depletion was confirmed in a nude mouse model. In conclusion, our findings indicate that BMI1 plays an important role in the late progression of pancreatic cancer and may represent a novel therapeutic target for the treatment of pancreatic cancer.