A central mediator of a wide host of target genes, the nuclear factor-κB (NF-κB) family of transcription factors, has emerged as a molecular target in cancer and diseases associated with bone destruction. To evaluate how NF-κB signaling in tumor cells regulates processes associated with osteolytic bone tumor burden, we stably infected the bone-seeking MDA-MB-231 breast cancer cell line with a dominant-negative mutant IκB that prevents phosphorylation of IκBα and associated nuclear translocation of NF-κB. Blockade of NF-κB signaling in MDA-MB-231 cells by the mutant IκB decreased in vitro cell proliferation, expression of the proinflammatory, bone-resorbing cytokine interleukin-6, and in vitro bone resorption by tumor/osteoclast cocultures while reciprocally up-regulating production of the proapoptotic enzyme caspase-3. Suppression of NF-κB transcription in these breast cancer cells also reduced incidence of in vivo tumor-mediated osteolysis after intratibial injection of tumor cells in female athymic nude mice. Immunohistochemistry showed that the cancerous lesions formed in bone by MDA-MB-231 cells express both interleukin-6 and the p65 subunit of NF-κB at the bone-tumor interface. NF-κB signaling in breast cancer cells therefore promotes bone tumor burden and tumor-mediated osteolysis through combined control of tumor proliferation, cell survival, and bone resorption. These findings imply that NF-κB and its associated genes may be relevant therapeutic targets in osteolytic tumor burden.