Apurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome, inhibit transcription and block replication. The primary enzyme to repair AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the Base Excision Repair (BER) pathway. Although the mechanism by which APE1 repairs AP sites in vitro has been extensively investigated, it is largely unknown how APE1 repairs AP sites in cells. Here, we show that APE1 is acetylated (AcAPE1) after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable Lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of positive charges of Lysine residues in the N-terminal domain of APE1 induces a conformation change; this in turn enhances the AP-endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Thus mammalian cells, unlike yeast or Escherichia coli, require acetylation of APE1 for efficient repairing of AP sites and base damages in the genome. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity.