Cucurbitacin B, a natural triterpenoid is well-known for its strong anticancer activity, and recent studies showed that the compound inhibits JAK/STAT3 pathway. In this study, we demonstrate for the first time that cucurbitacin B is also a potent inhibitor of NF-kB activation. Our results showed that cucurbitacin B inhibited TNF-a-induced expression of NF-kB reporter gene and NF-kB target genes in a dose-dependent manner, however, it did not prevent either stimuli-induced degradation of IkBa or nuclear translocation and DNA-binding activity of NF-kB. On the other hand, cucurbitacin B dose-dependently suppressed not only NF-kB activation induced by overexpression of RelA/p65 but also transactivation activity of RelA/p65 subunit of NF-kB. Consistently, treatment of HeLa cells with the compound significantly suppressed TNF-a-induced activation of Akt and phosphorylation of Ser536 in RelA/p65, which is required for transactivation activity. Consequently, cucurbitacin B inhibited TNF-a-induced expression of NF-kB-dependent anti-apoptotic proteins such as c-IAP1, c-IAP2, XIAP, TRAF1, and TRAF2 and sensitized TNF-a-induced cell death. Taken together, our results demonstrated that cucurbitacin B could be served as a valuable candidate for the intervention of NF-kB-dependent pathological condition such as cancer.