Xanthohumol (XN), a prenylated chalcone isolated from hop plant, exhibits anti-inflammatory, antiproliferative, and antiangiogenic properties through an undefined mechanism. Whether examined by intracellular esterase activity, phosphatidylserine externalization, DNA strand breaks, or caspase activation, we found that XN potentiated tumor necrosis factor-induced apoptosis in leukemia and myeloma cells. This enhancement of apoptosis correlated with down-regulation of nuclear factor-kappaB (NF-kappaB) survivin, bcl-xL, XIAP, cIAP1, cIAP2, cylin D1, and c-myc. XN down-regulated both constitutive and inducible NF-kappaB activation, inhibition of phosphorylation and degradation of IkappaBalpha, suppression of p65 nuclear translocation, and NF-kappaB-dependent reporter gene transcription. XN directly inhibited tumor necrosis factor-induced IkappaBalpha kinase (IKK) activation and a reducing agent abolished this inhibition, indicating the role of cysteine residue. XN had no effect on the IKK activity when cysteine residue 179 of IKK was mutated to alanine. XN also directly inhibited binding of p65 to DNA, a reducing agent reversed this effect, and mutation of cysteine residue 38 to serine of p65 abolished this effect. Thus, our results show that modification of cysteine residues of IKK and p65 by XN leads to inhibition of the NF-kappaB activation pathway, suppression of antiapoptotic gene products, and potentiation of apoptosis in leukemia cells.