Structure Maintenance of Chromosome 1 (SMC1) is phosphorylated by ATM in response to ionizing radiation (IR) to activate intra-S phase checkpoint. A role of CK2 in DNA damage response has been implicated in many previous works, but the molecular mechanism for its activation is not clear. In the present work, we report that SMC3 is phosphorylated at S1067 and S1083 in vivo. S1083 phosphorylation is IR inducible, depends on ATM and NBS1, and is required for intra-S phase checkpoint. Interestingly, S1067 phosphorylation is constitutive and is not induced by IR, but also affects intra-S phase checkpoint. Phosphorylation of S1083 is weakened in cells expressing S1067A mutant, suggesting interplay between S1067 and S1083 phosphorylation in DNA damage response. Consistently, siRNA knockdown of CK2 leads to attenuated phosphorylation of S1067 as well as intra-S phase checkpoint defect. Our data provide evidence that phosphorylation of a core cohesin subunit SMC3 by ATM plays important role in DNA damage response and suggest that a constitutive phosphorylation by CK2 may affects intra-S phase checkpoint by modulating SMC3 phosphorylation by ATM.