Rel/nuclear factor-κB (NF-κB) transcription factors control a variety of cellular processes, such as cell growth and apoptosis, and are continually activated in many human diseases, including chronic inflammatory diseases and cancer. Jesterone dimer (JD) is a synthetic derivative of the natural fungal metabolite jesterone, and JD has previously been shown to be cytotoxic in select tumor cell lines. In this report, we demonstrate that JD is a potent inhibitor of the activation of transcription factor NF-κB. Namely, JD inhibits tumor necrosis factor-α-induced activation of NF-κB in mouse 3T3 and human HeLa cells. JD seems to block the induction of the NF-κB pathway by inhibiting the inhibitor of κB kinase (IKK); that is, treatment of cells with JD blocks phosphorylation of IκBα, inhibits the activity of a constitutively active form of the IKKβ catalytic subunit, and converts IKKβ to stable high molecular mass forms. Like JD, a JD-related epoxyquinoid (isotorreyanic acid) inhibits activation of NF-κB at 20 μM, whereas several other epoxyquinoids that are related to JD, including its parent compound jesterone, do not block activation of NF-κB at this concentration. Finally, JD inhibits both proliferation and DNA binding by REL-containing complexes in the human lymphoma SUDHL-4 cell line, and JD activates caspase-3 activity in these cells. In summary, these results suggest that JD induces apoptosis in tumor cells through a mechanism that involves the inhibition of Rel/NF-κB activity and demonstrate the usefulness of assessing the bioactivity of synthetic derivatives of natural products.