We describe a novel regulatory mechanism for DNA polymerase β (Polβ), a protein involved in DNA base excision repair (BER). Polβ colocalized in vivo and formed a complex with the transcriptional coactivator p300. p300 interacted with Polβ through distinct domains and acetylated Polβ in vitro. Polβ acetylation was furthermore observed in vivo. Lysine 72 of Polβ was identified as the main target for acetylation by p300. Interestingly, acetylated Polβ showed a severely reduced ability to participate in a reconstituted BER assay. This was due to an impairment of the dRP-lyase activity of Polβ. Acetylation of Polβ thus acts as an intranuclear regulatory mechanism and implies that p300 plays a critical regulatory role in BER.