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The latent membrane protein 1 (LMP1) of Epstein-Barr virus causes cellular transformation and activates several intracellular signals, including NF-kappaB and c-Jun N-terminal kinase. Using yeast two-hybrid screening with the LMP1 C-terminal sequence as bait, we demonstrate that BRAM1 (bone morphogenetic protein receptor-associated molecule 1) is an LMP1-interacting protein. BRAM1 associates with LMP1, both in vitro and in vivo, as revealed by confocal microscopy, glutathione S-transferase pull-down, and co-immunoprecipitation assays. This association mainly involves the C-terminal half of BRAM1 comprising the MYND domain and the CTAR2 region of LMP1, which is critical in LMP1-mediated signaling pathways. We show that BRAM1 interferes with LMP1-mediated NF-kappaB activation but not the JNK signaling pathway. Because the CTAR2 region interacts with the tumor necrosis factor (TNF-alpha receptor-associated death domain protein, it is interesting to find that BRAM1 also interferes with NF-kappaB activation mediated by TNF-alpha. BRAM1 interferes LMP1-mediated and TNF-alpha-induced NF-kappaB activation by targeting IkappaBalpha molecules. Moreover, BRAM1 inhibits the resistance of LMP1-expressing cells to TNF-alpha-induced cytotoxicity. We therefore propose that the BRAM1 molecule associates with LMP1 and functions as a negative regulator of LMP1-mediated biological functions.