Acetylation of the RelA subunit of NF-κB, especially at lysine-310, is critical for the transcriptional activation of NF-κB and the expression of inflammatory genes. In this study, we demonstrate that bromodomains of Brd4 bind to acetylated lysine-310. Brd4 enhances transcriptional activation of NF-κB and the expression of a subset of NF-κB-responsive inflammatory genes in an acetylated lysine-310-dependent manner. Bromodomains of Brd4 and acetylated lysine-310 of RelA are both required for the mutual interaction and coactivation function of Brd4. Finally, we demonstrate that Brd4 further recruits CDK9 to phosphorylate C-terminal domain of RNA polymerase II and facilitate the transcription of NF-κB-dependent inflammatory genes. Our results identify Brd4 as a novel coactivator of NF-κB through specifically binding to acetylated lysine-310 of RelA. In addition, these studies reveal a mechanism by which acetylated RelA stimulates the transcriptional activity of NF-κB and the NF-κB-dependent inflammatory response.