Pifithrin-α (PFT-α) was shown to specifically block transcriptional activity of the tumor suppressor p53 and was therefore proposed to be useful in preventing the severe side effects often associated with chemo-and radiotherapy. We report here that although PFT-α efficiently protected different cell types from DNA damage-induced apoptosis, it mediated this effect regardless of the presence or absence of p53. Interestingly, PFT-α blocked the apoptosome-mediated processing and activation of caspase-9 and-3 without interfering with the activation of mitochondria. Neither the DNA damage-induced activation of Bax or Bak nor the loss of the mitochondrial membrane potential or the final release of cytochrome c were inhibited by this compound. Instead, the ability of PFT-α to protect p53-deficient cells from DNA damage-induced caspase activation and apoptosis was greatly diminished after siRNA-mediated downregulation of cyclin-D1 expression. In contrast, downregulation of other proteins involved in cell-cycle progression, such as the retinoblastoma protein, cyclin D3, as well as the cyclin-dependent kinases, 2, 4 and 6, could not abolish this protection. Thus, our data show that PFT-α protects cells from DNA damage-induced apoptosis also by a p53-independent mechanism that takes place downstream of mitochondria and that might involve cyclin D1.