DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-κB), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-κB dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-κB-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-κB-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-κB-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-κB transcriptional activity. J. Cell. Biochem. 106: 296–305, 2009. © 2008 Wiley-Liss, Inc.