DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for beta-TrCP (beta-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.