Abstract： Objective To investigate the effect and mechanism of NF-kB P65 gene silencing by small interference RNA on the apoptosis of human pancreatic cancer cells induced by gemcitabine in vitro and in vivo. Methods Human pancreatic cancer cells ( BxPC-3 and PANC-1 ) were cultured and respectively divided into five groups; blank control group,negative control siRNA group,gemcitabine group, NF-κB P65 siRNA group and gemcitabine + P65 siRNA group. The ability of cell proliferation was analyzed by MTT; the expression of NF-κB P65 and the apoptosis related proteins were examined by Westrn blot assay; the apoptosis was evaluated by the flow cytometry and laser scanning confocal microscopy analysis stained with Annexin V-FITC/PI; the DNA binding activity of NF-κB was examined by electrophoretic mobility shift assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors.The tumor volume was monitored and TUNEL assay was used to assess the apoptosis index in tumor tissue after treatment Results At 72 h after transfection, the combination with gemcitabine and p65 siRNA significantly decreased the cell viability index (P <0. 05) ,and down-regulated the expression of Bcl-2 and procaspase-3 and up-regulated the expression of Bax compared with other groups. The combinded treatment significantly increased the rate of apoptosis compared with other groups (P < 0. 05). EMSA assay indicated that the DNA binding activity of NF-κB significantly decreased in NF-κB P65 siRNA group and gemcitabine + P65 siRNA group compared with Control group. The combinded therapy inhibited the growth of pancreatic xenograft tumors by apoptosis induction in nude mice (P < 0. 01 ). Conclusions The effect of gemcitabine inducing cell apoptosis may be potentiated through inhibiting the DNA binding activity of NF-κBand regulating the expression of apoptosis related proteins by NF-?B P65 siRNA, which can activate the mitochondria apoptosis pathway in pancreatic cancer in vitro and in vivo.