Robust activation of poly(ADP-ribose) polymerase-1 (PARP) by oxidative stress has been implicated as a major cause of caspase-independent myocyte cell death contributing to heart failure. Here, we show that depletion of myocyte NAD levels and the subsequent reduction of Sir2α deacetylase activity are the sequential steps contributing to PARP-mediated myocyte cell death. In both failing hearts and cultured cardiac myocytes, the increased activity of PARP was associated with depletion of cellular NAD levels and reduced Sir2α deacetylase activity. Myocyte cell death induced by PARP activation was prevented by repletion of cellular NAD levels either by adding NAD directly to the culture medium or by overexpressing NAD biosynthetic enzymes. The beneficial effect of NAD repletion was seen, however, only when Sir2α was intact. Knocking down Sir2α levels by small interfering RNA eliminated this benefit, indicating that Sir2α is a downstream target of NAD replenishment leading to cell protection. NAD repletion also prevented loss of the transcriptional regulatory activity of the Sir2α catalytic core domain resulting from PARP activation. We also show that PARP activation and the concomitant reduction of Sir2α activity in failing hearts regulate the post-translational acetylation of p53. These data demonstrate that, in stressed cardiac myocytes, depletion of cellular NAD levels forms a link between PARP activation and reduced Sir2α deacetylase activity, contributing to myocyte cell death during heart failure.