Benzoxathiole derivatives have been used in the treatment of acne and have shown cytostatic, antipsoriatic, and antibacterial properties. However, little is known about the molecular basis for these pharmacological properties, although nuclear factor (NF)-kappaB activation is closely linked to inflammation and cell proliferation. Here, we demonstrate that the novel small-molecule benzoxathiole 6,6-dimethyl-2-(phenylimino)-6,7-dihydro-5H-benzo-[1,3]oxathiol-4-one (BOT-64) inhibits NF-kappaB activation with an IC(50) value of 1 muM by blocking inhibitory kappaB(IkappaB) kinase beta (IKKbeta), and suppresses NF-kappaB-regulated expression of inflammatory genes in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. BOT-64 inhibits IKKbeta-mediated IkappaBalpha phosphorylation in LPS-activated macrophages, resulting in sequential prevention of downstream events, including proteolytic degradation of IkappaBalpha, DNA binding ability, and transcriptional activity of NF-kappaB. BOT-64 inhibits LPS-inducible IKKbeta activity in the cells and catalytic activity of highly purified IKKbeta. Moreover, the effect of BOT-64 on cell-free IKKbeta was abolished by substitution of Ser-177 and Ser-181 residues in the activation loop of IKKbeta to glutamic acid residues, indicating a direct interaction site of benzoxathiole. BOT-64 attenuates NF-kappaB-regulated expression of inflammatory genes such as inducible nitric-oxide synthase, cyclooxygenase-2, tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 in LPS-activated or expression vector IKKbeta-transfected macrophages. Furthermore, BOT-64 dose-dependently increases the survival rates of endotoxin LPS-shocked mice.