The Ku heterodimer facilitates the regulation of DNA repair, DNA replication, cell cycle, and telomere maintenance. The plant hormone abscisic acid (ABA) is a plant growth inhibitor. This study investigates how Arabidopsis thaliana Ku (AtKu) genes are regulated by ABA in 3-week-old seedlings. First, beta-Glucuronidase assay and real time quantitative PCR analysis results indicate that ABA represses the AtKu gene in a time- and concentration-dependent manner. However, adding of ABA biosynthesis inhibitors, fluride and tungate, did not eliminate AtKu suppression. Moreover, analysis of inhibitor treatments and ABA-responsive mutants suggested that AtKu repression by ABA was mediated through the pathway of extracellular Ca(2+), phospholipase D alpha, p38-type mitogen-activated protein kinase (MAPK), MAPK6 and ABA transcription factors, ABI3 and ABI5. Finally, no cross-talk in modulating AtKu gene expression existed between ABA and antagonist hormones (auxins and gibberellic acid).