AbstractCyclin A1 plays a critical role in hematopoietic malignancies, notably, acute myeloid leukemia. The molecular mechanisms of cyclin A1 action are incompletely understood. Here, we show that cyclin A1 functions are mediated by the retinoblastoma and the Ku70 pathway. High levels of cyclin A1 and the associated CDK2 kinase activity were associated with increasing levels of phosphorylated retinoblastoma in vivo. UV irradiation induced a switch of the CDK2 towards cyclin A1, with accordance to changes in CDK2 kinase activity. The C‐terminus of cyclin A1 directly interacted with Ku70, and DNA binding activity of Ku70 was modulated by cyclin A1/CDK2 and phosphatase treatment. Cyclin A1‐deficiency induced by shRNA increased apoptosis that is induced by DNA damage and death receptor ligands. Taken together, these analyses demonstrate that cyclin A1 exerts antiapoptotic functions by interacting with retinoblastoma and Ku proteins in leukemia cells. © 2007 Wiley‐Liss, Inc.