The transcription factor E2F1 has pivotal roles in both cell proliferation and cell death, and is an important molecular target in cancer. Under proliferative conditions E2F1 induces the expression of genes that promote cell cycle progression, such as E2F2, whereas under proapoptotic conditions E2F1 induces expression of genes such as p73 that lead to apoptosis. The mechanism by which the apoptotic function of E2F1 is activated remains unclear, however. We now show that members of the E2F family are covalently conjugated with the ubiquitin-like modifier NEDD8. Overexpression of SENP8, a NEDD8-specific cysteine protease, resulted in deNEDDylation of E2F1 and promoted its transactivation activity at the p73 gene but not at the E2F2 gene. Knockdown of SENP8, on the other hand, attenuated p73 expression and apoptosis induced by E2F1 or by DNA damage. SENP8 also promoted the interaction between E2F1 and its cofactor Microcephalin 1, which is required for p73 induction. These results suggest that NEDDylation is a molecular trigger that modifies the target specificity of E2F1, and could have important implications for E2F1 regulation of apoptosis.