AbstractDEAD‐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.