Abstract: Objective RNA interference (RNAi) was employed to inhibit the expression of p65 and to evaluate the effects of NF-κB signaling pathway as a target for gene therapy in ESCC cell lines. Methods A fluorescein-labeled non-special siRNA was used to monitor efficiency in EC9706. EC9706 and Eca109 were transfected with 50 nM p65siRNA, p65 and cyclinD1 protein levels were determined using Western Blotting. After transfected with or without p65siRNA at 72h, cells were tested for nuclear activity of NF-κB using nuclear extracts by EMSA. ESCC cells were t reated with or without p65siRNA, 5-Fu or combinations thereof for 48 h. The cells were stained with PI and analyzed cell cycle by flow cytometry. Results 　A fluorescein-labeled non-special siRNA was used to monitor efficiency in EC9706, demonstrating nearly 90% t ransfection efficiency. The protein level of p65 and cyclinD1 decreased af ter t ransfected with p65siRNA at 72 h. And p65siRNA also suppressed the NF-κB-DNA-binding. p65siRNA-transfected cells showed an increase in the percentage of cells in the G₀ / G₁ phase and a decrease in the percentage of cells in the Sphase. While combination with 5-Fu, the result s were more prominence. Conclusion p65siRNA inhibit s the constitutively active NF-κB signaling pathway, and downregulat s the expression of cyclinD1. The st rong constitutive NF-κB signaling pathway in ESCC makes NF-κB a target for the development of novel therapeutic st rategies.