Evodiamine, an alkaloidal component extracted from the fruit of Evodiae fructus (Evodia rutaecarpa Benth., Rutaceae), exhibits antiproliferative, antimetastatic, and apoptotic activities through a poorly defined mechanism. Because several genes that regulate cellular proliferation, carcinogenesis, metastasis, and survival are regulated by nuclear factor-kappa B (NF-kappa B), we postulated that evodiamine mediates its activity by modulating NF-kappa B activation. In the present study, we investigated the effect of evodiamine on NF-kappa B and NF-kappa B-regulated gene expression activated by various carcinogens. We demonstrate that evodiamine was a highly potent inhibitor of NF-kappa B activation, and it abrogated both inducible and constitutive NF-kappa B activation. The inhibition corresponded with the sequential suppression of I kappa B alpha kinase activity, I kappa B alpha phosphorylation, I kappa B alpha degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Evodiamine also inhibited tumor necrosis factor (TNF)-induced Akt activation and its association with IKK. Suppression of Akt activation was specific, because it had no effect on JNK or p38 MAPK activation. Evodiamine also inhibited the NF-kappa B-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK but not that activated by the p65 subunit of NF-kappa B. NF-kappa B-regulated gene products such as Cyclin D1, c-Myc, COX-2, MMP-9, ICAM-1, MDR1, Survivin, XIAP, IAP1, IAP2, FLIP, Bcl-2, Bcl-x(L), and Bfl-1/A1 were all down-regulated by evodiamine. This down-regulation potentiated the apoptosis induced by cytokines and chemotherapeutic agents and suppressed TNF-induced invasive activity. Overall, our results indicated that evodiamine inhibits both constitutive and induced NF-kappa B activation and NF-kappa B-regulated gene expression and that this inhibition may provide a molecular basis for the ability of evodiamine to suppress proliferation, induce apoptosis, and inhibit metastasis.