Checkpoint kinase 2 (Chk2), a DNA damage-activated protein kinase, is phosphorylated at Thr-68 by ataxia telangiectasia mutated leading to its activation by phosphorylation at several additional sites. Using mass spectrometry we identified a new Chk2 phosphorylation site at Ser-456. We show that phosphorylation of Ser-456 plays a role in the regulation of Chk2 stability particularly after DNA damage. Mutation of Ser-456 to alanine results in hyperubiquitination of Chk2 and dramatically reduced Chk2 stability. Furthermore, cells expressing S456A Chk2 show a reduction in the apoptotic response to DNA damage. These findings suggest a mechanism for stabilization of Chk2 in response to DNA damage via phosphorylation at Ser-456 and proteasome-dependent turnover of Chk2 protein via dephosphorylation of the same residue.