Activating transcription factor 3 (ATF3) is a transcriptional repressor that is rapidly induced in cells exposed to a wide range of stress stimuli. To clarify the role of ATF3 in determining cell fate, we overexpressed it in human umbilical vein endothelial cells (HUVECs) by adenovirus-mediated gene transfer. ATF3 protected these cells from tumor necrosis factor (TNF)-alpha-induced apoptosis, as measured by flow cytometric analysis, trypan blue exclusion assay, and cleavage of procaspase 3 and poly(ADP-ribose) polymerase. Northern blot and nuclear run on assay showed that the transcription of tumor suppressor gene p53 was down-regulated in the ATF3-overexpressing cells. In the transient expression assay, ATF3 suppressed the p53 gene promoter activity through its specific binding to an atypical AP-1 element, PF-1 site, in the p53 gene promoter. Furthermore, the cell-protecting effect of ATF3 was remarkably reduced in p53-deficient cells. These results demonstrate that overexpression of ATF3 suppresses TNF-alpha-induced cell death of HUVECs, at least in part, through down-regulating the transcription of p53 gene. ATF3 may function as a cell survival factor of endothelial cells during vascular inflammation and atherogenesis.