DNA fragmentation is a hallmark of apoptosis. The tightly controlled activation of the apoptosis-specific endonucleases provides an effective means to ensure the removal of unwanted DNA and the timely completion of apoptosis. Over the past several years, crucial progress has been made in identifying the long-awaited apoptotic endonucleases, and their importance in tissue homeostasis is beginning to unfold. Here, we focus on the most recent discoveries about the functions and mechanisms of these endonucleases in the context of apoptosis. We also discuss consequences that defective DNA fragmentation might have for tissue homeostasis and disease development.