The cellular response to DNA double-strand break (DSB) occurs through an integrated sensing and signalling network that maintains genomic stability. Oestrogen (E2), among its many functions, is known to have a positive effect on global genomic DNA repair; however, the mechanism by which it functions is unclear. A central enzyme involved in DNA DSB repair in mammalian cells is the DNA-dependent protein kinase (DNA-PK). Here, we show that E2 enhances DNA-PK catalytic subunit (DNA-PKcs) promoter activity with subsequent transcriptional and translational upregulation of DNA-PKcs in a breast cancer cell line. We identify two potential E2 receptor-alpha (ERalpha)-binding sites in a region upstream from the DNA-PKcs initiation site. By using small interfering RNA and the specific E2 receptor antagonist ICI 182,780, we demonstrate that ERalpha knockdown reduces E2-induced upregulation of DNA-PKcs expression and activity in breast carcinoma cells. E2-induced DNA-PK transactivation results in an increased ability of the cells to repair DNA DSB. This previously unknown mechanism of DNA-PK regulation sheds new light on tumour biology and reveals new possibilities for the prevention and therapy of E2-sensitive proliferative diseases.