We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.