The Ku70/Ku80 heterodimer plays a crucial role in non-homologous end-joining during DNA repair, and is also involved in multiple cellular processes such as telomere maintenance, transcription, and apoptosis. In this study, we investigate the regulation of AtKu genes in higher plants. Promoters of the AtKu70 and AtKu80 were isolated from Arabidopsis and their activities characterised using GUS reporter constructs. AtKu promoter activities were relatively higher in hypocotyls and cotyledons upon germination and in stigma and siliques as well at their early developing stages. Furthermore, AtKu promoter activities could be enhanced by gibberellic acid, auxins, and jasmonic acid, but repressed by abscisic acid, salicylic acid, heat, drought and cold, respectively. Deletion analysis demonstrates minimal lengths of ~400 bp and 600 bp upstream of transcription start site for functional promoters of AtKu70 and AtKu80, respectively. Taken together, expressions of Ku genes are regulated both by developmental programs as well as by plant hormones and environmental stresses.