The transcription factor p53 plays a crucial role in coordinating the cellular response to various stresses. Therefore, p53 protein levels and activity need to be kept under tight control. We report here that diacylglycerol kinase ζ (DGKζ) binds to p53 and modulates its function both in the cytoplasm and nucleus. DGKζ, one of the DGK family that metabolizes a lipid second messenger diacylglycerol, localizes primarily to the nucleus in various cell types. Recently, reports have described that excitotoxic stress induces DGKζ nucleocytoplasmic translocation in hippocampal neurons. In this study, we found that cytoplasmic DGKζ attenuates p53-mediated cytotoxicity against doxorubicin-induced DNA damage by facilitating cytoplasmic anchoring and degradation of p53 through a ubiquitin–proteasome system. Concomitantly, decreased levels of nuclear DGKζ engender down-regulation of p53 transcriptional activity. Consistent with these in vitro cellular experiments, DGKζ-deficient brain exhibits high levels of p53 protein after kainate-induced seizures and even under normal conditions. These findings provide novel insights into the regulation of p53 function and suggest that DGKζ serves as a sentinel to control p53 function both during normal homeostasis and in stress responses.