The inducible transcription factor NF-kappaB regulates divergent signaling pathways including inflammatory response and cancer development. Selective inhibitors for NF-kappaB signaling are potentially useful for treatment of inflammation and cancer. NF-kappaB is canonically activated by preferential disposal of its inhibitory protein; IkappaB, which suppresses the nuclear translocation of NF-kappaB. IkappaBalpha (a major member of IkappaB family proteins) is phosphorylated with an IkappaB kinase (IKK) and subsequently polyubiquitylated by SCF(betaTrCP1) ubiquitin-ligase in the presence of E1 and E2 prior to proteasomal degradation. Here, we describe a novel inhibitor termed GS143, which suppressed IkappaBalpha ubiquitylation, but not IkappaBalpha phosphorylation, MDM2-directed p53 ubiquitylation, and proteasome activity in vitro. GS143 markedly suppressed the destruction of IkappaBalpha stimulated by TNFalpha and a set of downstream responses coupled to NF-kappaB signaling but not those of p53 and beta-catenin in vivo. Our results indicate that GS143 serves as an effective inhibitor of multiple pathways served by NF-kappaB signaling.