The cyclic AMP response element binding protein (CREB) initiates transcriptional responses to a wide variety of stimuli. CREB activation involves its phosphorylation on serine 133 (S133), which promotes interaction between the CREB kinase inducible domain (KID) and the KID-interacting (KIX) domain of the transcriptional coactivator, CREB binding protein (CBP). The KID also contains a highly conserved phosphorylation cluster, termed the ATM/CK cluster, that is processively phosphorylated in response to DNA damage by the coordinated actions of ataxia-telangiectasia-mutated (ATM) and casein kinases (CKs) 1 and 2. ATM/CK cluster phosphorylation attenuates CBP binding and CREB transcriptional activity . Paradoxically, it was recently reported that DNA damage activates CREB through homeodomain interacting protein kinase 2-dependent phosphorylation of S271 near the CREB bZIP DNA-binding domain. In this study we sought to further clarify DNA damage-dependent CREB phosphorylation as well as to explore the possibility that the ATM/CK cluster and S271 synergistically or antagonistically modulate CREB activity. We show that, rather than being induced by DNA damage, S270 and S271 of CREB co-phosphorylated in a CDK1-dependent manner during G2/M phase. Functionally, we show that phosphorylation of CREB on S270/271 during mitosis correlated with reduced CREB chromatin occupancy. Furthermore, CDK1-dependent phosphorylation of CREB in vitro inhibited its DNA-binding activity. The combined results suggest that CDK1-dependent phosphorylation of CREB on S270/271 facilitates its dissociation from chromatin during mitosis by reducing its intrinsic DNA-binding potential.