DFF45 is a subunit of the DNA fragmentation factor (DFF) that is cleaved by caspase‐3 during apoptosis. However, the mechanism by which DFF45 regulates apoptotic cell death remains poorly understood. Here we report the identification and characterization of two mammalian genes, CIDE‐A and CIDE‐B, encoding highly related proteins with homology to the N‐terminal region of DFF45. CIDE‐A and CIDE‐B were found to activate apoptosis in mammalian cells, which was inhibited by DFF45 but not by caspase inhibitors. Expression of CIDE‐A induced DNA fragmentation in 293T cells, which was inhibited by DFF45, further suggesting that DFF45 inhibits the apoptotic activities of CIDEs. In addition to mammalian CIDE‐A and CIDE‐B, we identified DREP‐1, a Drosophila melanogaster homolog of DFF45 that could inhibit CIDE‐A‐mediated apoptosis. Mutant analysis revealed that the C‐terminal region of CIDE‐A was necessary and sufficient for killing whereas the region with homology to DFF45 located in the N‐terminus was required for DFF45 to inhibit CIDE‐A‐induced apoptosis. CD95/Fas‐mediated apoptosis was enhanced by CIDEs but inhibited by DFF45. These studies suggest that DFF45 is evolutionarily conserved and implicate CIDEs as DFF45‐inhibitable effectors that promote cell death and DNA fragmentation.