To study the impact of coxsackievirus B3 2A protease on nuclear factor κB (NF-κB ), BHK cells were transfected with a plasmid pcDNA3.1-2A expressing CVB3 2A protease along with pGL3-NF-κB -promotor-luc. The activity of luciferase was detected to identify the relative effect of NF-κB upon tumor necrosis factor α (TNF-α) stimulation. The dimerization of NF-κB /p65 were also detected with Western blotting and translation of NF-κB /p65 was observed by immunofluorescence. The results showed that CVB3 2A protease not only inhibited the expression of luciferase activities from NF-κB /p65 promoter but also interfered the nuclear translocation of NF-κB /p65. CVB3 2A protease still inhibited the dimerization of NF-κB /p65. It is concluded that CVB3 protease 2A inhibits the activation of NF-κB pathway.