The early response gene IEX-1 plays a complex role in the regulation of apoptosis. Depending on the cellular context and the apoptotic stimulus, IEX-1 is capable to either enhance or suppress apoptosis. To further dissect the molecular mechanisms involved in the modulation of apoptosis by IEX-1, we analysed the molecular crosstalk between IEX-1 and the NF-κB pathway. Using GST-pulldown assays, a direct interaction of IEX-1 with the C-terminal region of the subunit RelA/p65 harbouring the transactivation domain of the NF-κB transcription factor was shown. This interaction negatively regulates RelA/p65 dependent transactivation as shown by GAL4-and luciferase assay and was confirmed for the endogenous proteins by co-immunoprecipitation experiments. Using deletion constructs, we were able to map the C-terminal region of IEX-1 as the critical determinant of the interaction with RelA/p65. We could further show, that IEX-1 mediated NF-κB inhibition accounts for the reduced expression of the anti-apoptotic NF-κB target genes Bcl-2, Bcl-xL, cIAP1 and cIAP2, thereby sensitizing cells for apoptotic stimuli. Finally, ChIP-assays revealed that IEX-1 associates with the promoter of these genes. Altogether, our findings suggest a critical role of IEX-1 in the NF-κB dependent regulation of apoptotic responses.