NEEP21, also designated D4S234E or NSG1, is an endosomal protein expressed in neuronal cells under normal conditions. Here, we report that NEEP21 is a direct transcriptional target gene of the tumor suppressor p53. NEEP21 expression is inducible in non-neuronal human cancer cell lines by exposure to adriamycin, hydrogen peroxide, UV and γ-ray in a p53-dependent manner. Chromatin immunoprecipitation assay indicated that a potential p53-binding site (p53BS) is located in intron 1 of the NEEP21 gene. A reporter assay confirmed that p53BS has p53-responsive activity. The heterologous luciferase gene containing p53BS is also transactivated by p73-β and p63-γ. The introduction of the NEEP21 gene into various cancer cell lines suppressed cell growth. Infection with an adenovirus vector containing NEEP21 induced apoptotic cell death via caspase-3 activation in many cancer cell lines. The expression of NEEP21 mRNA was remarkably induced by γ-ray irradiation in the spleen of p53+/+ mice but not in that of p53-/- mice. These results suggest that NEEP21 may play a critical role in apoptosis as a mediator of p53.