DNA topoisomerase II is an essential nuclear enzyme that modulates DNA processes by altering the topological state of double-stranded DNA. This enzyme is required for chromosome condensation and segregation; however, the regulatory mechanism of its activation is largely unknown. Here we demonstrate that topoisomerase IIα is activated in response to genotoxic stress. Concomitant with the activation, the expression of topoisomerase IIα is increased following DNA damage. The results also demonstrate that the proapoptotic kinase protein kinase C δ (PKCδ) interacts with topoisomerase IIα. This association is in an S-phase-specific manner and is required for stabilization and catalytic activation of topoisomerase IIα in response to DNA damage. Conversely, inhibition of PKCδ activity attenuates DNA damage-induced activation of topoisomerase IIα. Finally, aberrant activation of topoisomerase IIα by PKCδ is associated with induction of apoptosis upon exposure to genotoxic agents. These findings indicate that PKCδ regulates topoisomerase IIα and thereby cell fate in the genotoxic stress response.