Activating transcription factor 3 (ATF3) is induced by inflammatory responses, cell death, cytokines, and oxidative stress conditions. ATF3 is a negative regulator in the Toll-like receptor 4 signalling pathway. The principal molecule in this pathway is nuclear factor κB (NF-κB) that translocates into the nucleus to initiate the transcription of inflammatory mediators. However, scarce data are available regarding the interaction of ATF3 and p65, a part of the NF-κB dimer. Therefore, we studied the mechanism of regulation of p65 by ATF3 in RAW 264.7 cells. First, LPS-mediated NF-κB activation was confirmed, and then the direct interaction of ATF3 and p65 was observed through immunoprecipitation (IP). The presence of histone deacetylase 1 (HDAC1) was also detected in the complex. In ATF3 deficient cells, NF-κB activity was up-regulated and HDAC1 was not detected by IP. These observations suggest that p65 is attenuated by ATF3 such that ATF3 recruits HDAC1 to the ATF3/p65 complex and facilitates the deacetylation of p65. Likewise, inflammatory response genes were induced by translocated NF-κB in ATF3-deficient cells. Cumulatively, we uncovered a novel mechanism for the negative regulation of NF-κB by ATF3 via direct interaction with p65.