Aberrant constitutive expression of the NF-kappaB c-Rel and RelA subunits in breast cancer cells was shown to promote their survival. Recently, we demonstrated that aggressive breast cancers constitutively express high levels of the RelB subunit, which promotes their more invasive phenotype via induction of the BCL2 gene. As these cancers are frequently resistant to therapy, here we tested the hypothesis that RelB promotes their survival. High RelB expressing Hs578T and MDA-MB-231 breast cancer cells were more resistant to gamma-radiation than MCF7 and ZR-75 cells, which express lower RelB levels. Knockdown of RelB in Hs578T led to decreased survival in response to gamma-irradiation, while conversely ectopic expression of RelB in MCF7 cells protected these cells from radiation. Similar data were obtained upon treatment of Hs578T or MCF7 cells with the chemotherapeutic agent doxorubicin. High serum levels of 25-hydroxyvitamin D are associated with decreased breast cancer risk and mortality, although, the mechanisms of its protective actions have not been fully elucidated. Treatment of Hs578T and Her-2/neu-driven NF639 cells with 1,25-dihydroxyvitamin D3 decreased RelB/RELB gene expression and levels of pro-survival targets Survivin, MnSOD and Bcl-2, while increasing their sensitivity to gamma-irradiation. Thus, RelB, which promotes survival and a more highly invasive phenotype of breast cancer cells, is a target of 1,25-dihydroxyvitamin D3, providing one mechanism for the observed protective role of 25-hydroxyvitamin D in patients with breast cancer.