Objective To study the effect of knocking down B-cell specific moloney leukemia virus insertion site 1 (Bmi-1) on cell proliferation,the methylation of p16 and the expression of p16.Methods Antisense Bmi-1 vector was constructed and transfected into human pancreatic cancer cell line (PANC-1) cells,and the efficiency of transfection was evaluated by fluorescence microscope and Western blotting.Cell cycle and apoptosis were examined by using flow cytometry.The expression of p16 and the methylation of p16 promotor were detected by using Western blotting,real-time polymerase chain reaction (PCR) and methylation-specific PCR,respectively.Results Antisense Bmi-1 vector was successfully constructed,and the transfection efficiency was about 90%.The percentage of G0G1 cells was increased from 40.52% to 60.48% (P < 0.05) and apoptosis rate increased from 7.87% to 21.67% (P < 0.05).The expression of Bmi-1 protein was reduced from 318.54 ± 1.21 to 175.39 ±0.73 (P <0.05),the expression levels of p16 RNA and protein in the transfected cells were increased to 8.621 ± 0.310 and 304.12 ±0.76 respectively (P < 0.05),and the promotor methylation of p16 was declined (P < 0.05).Conclusion Antisense Bmi-1 vector may knock down the expression of Bmi-1 in PANC-1 cells and could inhibit the proliferation of cells.The expression of p16 is possibly regulated by Bmi-1 via promoting the methvlation of o16 oromotor. Key words: Pancreatic carcinoma; Bmi-1 ; Methylation; p16