In a number of contexts, and particularly in response to cellular stress, stimulation of the NF-kappaB (NF-κB) pathway promotes apoptosis. One mechanism underlying this pro-apoptotic activity is nucleolar sequestration of RelA, which is reported to cause cell death by repressing NF-κB-driven transcription. Here, we identify a novel and distinct nucleolar activity of RelA that induces apoptosis. We demonstrate, using a viral nucleolar localization signal (NoLS)-RelA fusion protein, that direct targeting of RelA to the nucleolus mediates apoptosis, independent of NF-κB transcriptional activity. We demonstrate a requirement for nucleophosmin (NPM, B23.1) in this apoptotic effect, and the apoptotic effect of stress-induced nucleolar RelA. We show by multiple approaches that nucleolar translocation of RelA is causally involved in the relocalization of NPM from the nucleolus to the cytoplasm and that RelA-induced cytoplasmic NPM mediates apoptosis by facilitating the mitochondrial accumulation of BAX. These data uncover a novel stress-response pathway and mechanism by which RelA promotes apoptosis, independent of its effects on NF-κB transcriptional activity. These findings are relevant to the design of novel anticancer agents that target RelA to this compartment.