Exposure of colorectal cancer (CRC) cells to ionizing radiation results in a cell-cycle arrest in G1 and G2. The G1 arrest is due to p53-mediated induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/SDI1, but the basis for the G2 arrest is unknown. Through a quantitative analysis of gene expression patterns in CRC cell lines, we have discovered that 14-3-3sigma is strongly induced by gamma irradiation and other DNA-damaging agents. The induction of 14-3-3sigma is mediated by a p53-responsive element located 1.8 kb upstream of its transcription start site. Exogenous introduction of 14-3-3sigma into cycling cells results in a G2 arrest. As the fission yeast 14-3-3 homologs rad24 and rad25 mediate similar checkpoint effects, these results document a molecular mechanism for G2/M control that is conserved throughout eukaryotic evolution and regulated in human cells by p53.