The nuclear factor-kappa B (NF-κB) is a pleiotropic transcription factor regulating the expression of genes involved in various biological processes including the immune response and inflammation. Lamprey is regarded as a key species to provide meaningful clues for understanding the evolution of immune system; nevertheless, no information about lamprey NF-κB is reported. Thus, we have characterized a NF-κB homolog in lamprey (lj-NF-κB) for the deeper understanding of the role it played in lamprey immune system. The sequence and 3D structure analyses demonstrate that lj-NF-κB contained a Rel homology domain (RHD) and seven ankyrin repeats domains (ANKs), which would exhibit functional similarities to NF-κB superfamily proteins. This hypothesis was further proved by experiments. We found that the RHD of lj-NF-κB could interact with a mammalian κB response element, translocate to the nucleus to modulate gene (IL-6, IL-1β and TNF-α) expression, and the nuclear localization signals (NLS) was essential for the nuclear translocation. Furthermore, the ANKs of lj-NF-κB are the inhibition signal for the RHD of lj-NF-κB. The present results allow us to surmise that the lj-NF-κB should play a key role in immune response of lamprey, and the function of NF-κB has been maintained during evolution.