Objective: To investigate the effect of gene silencing of DNMT1 and DNMT3b on methylation in the promoter region of p16 and RASSF1A gene in pancreatic carcinoma BxPC-3 cells. Methods: The pancreatic carcinoma BxPC-3 cells were divided into five groups, which included the DNMT1 interference group (transfected with DNMT1-siRNA), DNMT3b interference group (transfected with DNMT3b-siRNA), double interference group (transfected with DNMT1+DNMT3b-siRNA), negative control group (transfected negative-siRNA) and blank control group (transfected with lipofectamine). Forty-eight hours after transfection, the mRNA and protein expression of DNMT1 and DNMT3b were analyzed by real-time RT-PCR and Western blot, and the methylation status in the promoter region of p16 and RASSF1A gene was detected with methylation-specific PCR (MSP). Results: Compared with the negative or blank control group, the mRNA and protein expression levels of the targeted genes were all markedly down-regulated in each interference group (all P<0.01). The methylation status of p16 and RASSF1A gene was positive in both the blank and negative control group; the p16 gene was unmethylated and RASSF1A gene was partially methylated in DNMT1 interference group and double interference group; the p16 gene was partially methylated and RASSF1A gene showed methylation-positive state in DNMT3b interference group. Conclusion: DNMT1 interference has better effect than DNMT3b interference on demethylation in BxPC-3 cells, and the double knockdown of DNMT1 and DNMT3b exerts no synergistic action. DNMT1 could serve as an effective target for the demethylation therapy of pancreatic carcinoma.