NF-κB transcription factors play important roles in immune responses and the development of the immune system. Many aspects of NF-κB signaling differ significantly among distinct species, although many similarities in signaling exist in flies and humans. Thus, to understand the functional refinement of the NF-κB cascade from invertebrates to vertebrates, the Rel and NF-κB proteins, identified as bbtRel and bbtp105, were characterized in a basal chordate amphioxus. Consistent with the sequence similarities, bbtRel was found to interact with a mammalian κB response element, to move into the nucleus when activated, and to be inhibited by the NF-κB-specific inhibitor helenalin. Similar to the other class I members, bbtp105 could be cleaved into the mature form p58. Such endoproteolysis depends on the GRR sequence and requires both protease degradation and caspase 8 cleavage. Furthermore, we found that bbtIκB and the unprocessed bbtp105 can inhibit the transcriptional activity of bbtRel, whereas bbtp58 forms homodimers or heterodimers with bbtRel to create a mature NF-κB complex. Finally, we found that the survival rate and the expression of bbtIκB and TNF-α-like genes were decreased when adult amphioxus were treated with helanalin before immune challenge, suggesting the archaic roles for NF-κB signaling in innate immune responses in a basal chordate. The presence of the NF-κB-IκB cascade in amphioxus indicates that it is a significant feature linking invertebrates to vertebrates and is refined in vertebrates through the expansion and divergence of genes involved in the cascade.