DNA-dependent Protein Kinase (DNA-PK) coordinates the repair of double-strand breaks through non-homologous end joining, the dominant repair pathway in mammalian cells. Breaks can also be resolved through homologous recombination during S/G2 cell cycle phases, initiated by the Mre11-Rad50-Nbs1 (MRN) complex and CtIP-mediated resection of 5’ strands. The functions of DNA-PK are considered to be end-joining specific, but here we demonstrate that human DNA-PK also plays an important role in the processing of DNA double-strand breaks. Using ensemble biochemistry and single-molecule approaches, we show that the MRN complex in cooperation with CtIP is stimulated by DNA-PK to perform efficient endonucleolytic processing of DNA ends in physiological conditions. This activity requires both CDK and ATR phosphorylation of CtIP. These unexpected results could explain the absence of DNA-PK deletion mutations in the human population, as homologous recombination is an essential process in mammals.