Posttranslational modifications of the RelA subunit of NF-kappaB, including acetylation and methylation, play a key role in controlling the strength and duration of its nuclear activity. Whether these modifications are functionally linked is largely unknown. Here, we show that the acetylation of lysine 310 of RelA impairs the Set9-mediated methylation of lysines 314 and 315, which is important for the ubiquitination and degradation of chromatin-associated RelA. Abolishing the acetylation of lysine 310 either by the deacetylase SIRT1 or by mutating lysine 310 to arginine enhances methylation. Conversely, enhancing the acetylation of lysine 310 by depleting SIRT1 or by replacing lysine 310 with acetyl-mimetic glutamine inhibits methylation, thereby decreasing ubiquitination, prolonging the stability of chromatin-associated RelA, and enhancing the transcriptional activity of NF-kappaB. The acetylation of lysine 310 of RelA interferes with its interaction with Set9. Based on structural modeling of the SET domain of Set9 with RelA, we propose that the positive charge of lysine 310 is critical for the binding of RelA to a negatively charged "exosite" within the SET domain of Set9. Together, these findings demonstrate for the first time an interplay between RelA acetylation and methylation and also provide a novel mechanism for the regulation of lysine methylation by acetylation.