Histone deacetylase (HDAC) inhibitors have been used to promote neuronal survival and ameliorate neurological dysfunction in a host of neurodegenerative disease models. The precise molecular mechanisms whereby HDAC inhibitors prevent neuronal death are currently the focus of intensive research. Here we demonstrate that HDAC inhibition prevents DNA damage-induced neurodegeneration by modifying the acetylation pattern of the tumor suppressor p53, which decreases its DNA-binding and transcriptional activation of target genes. Specifically, we identify that acetylation at K382 and K381 prevents p53 from associating with the pro-apoptotic PUMA gene promoter, activating transcription, and inducing apoptosis in mouse primary cortical neurons. Paradoxically, acetylation of p53 at the same lysines in various cancer cell lines leads to the induction of PUMA expression and death. Together, our data provide a molecular understanding of the specific outcomes of HDAC inhibition and suggest that strategies aimed at enhancing p53 acetylation at K381 and K382 might be therapeutically viable for capturing the beneficial effects in the CNS, without compromising tumor suppression.