Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF-kappaB signaling pathways through the two C-terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1-induced c-Jun N-terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor-associated factor 6. In the present study, our manipulation of BS69 expression clearly indicates that BS69 negatively regulates LMP1-mediated NF-kappaB activation and up-regulates IL-6 mRNA expression and IkappaB degradation. Our immunoprecipitation experiments suggest that BS69 decreases complex formation between LMP1 and TNFR-associated death domain protein (TRADD).