The MYST acetyltransferase HBO1 is implicated in the regulation of DNA replication and activities of transcription factors such as the androgen receptor. Since the androgen receptor and NF-kappaB transcription factors crossmodulate their transcriptional activity, we investigated whether HBO1 regulates NF-kappaB signaling. Here, we report that in 293T cells HBO1 reduced dose-dependently NF-kappaB activity stimulated by TNFalpha, or by overexpressing p65/RelA, RelB, or cRel. Mutational analysis showed that the N-terminal serine-rich region of HBO1 but not the acetyltransferase function was required for inhibition. Electrophoretic mobility-shift assays demonstrated that HBO1 was neither perturbing the formation of p65/RelA DNA complexes nor binding itself to the kappaB consensus sequence or to p65/RelA, suggesting that HBO1 reduced NF-kappaB activity by squelching a cofactor. These data establish a novel function for HBO1 showing that it reduced NF-kappaB activity by sequestrating an essential coactivator from the NF-kappaB transcriptional complex.