Normal intestinal mucosal growth requires polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. Our previous studies have shown that polyamine depletion stabilizes p53 resulting in inhibition of intestinal epithelial cell (IEC) proliferation, but the exact down-stream targets of induced p53 are still unclear. The NDRG1 (N-Myc down-regulated gene-1) gene encodes a growth-related protein and its transcription can be induced in response to stress. The current study tests the hypothesis that induced p53 inhibits IEC proliferation by up-regulating NDRG1 expression following polyamine depletion. Depletion of cellular polyamines by a-difluoromethylornithine, the specific inhibitor of polyamine biosynthesis, not only induced p53 expression but also increased NDRG1 transcription as indicated by induction of the NDRG1-promoter activity and increased levels of NDRG1 mRNA and protein, all of which were prevented by using specific p53 siRNA and in cells with a targeted deletion of p53. Consistently, polyamine depletion-induced activation of the NDRG1-promoter was decreased when p53-binding sites within the NDRG1 proximal promoter region were deleted. Ectopic expression of the wild-type NDRG1 gene inhibited DNA synthesis and decreased final cell numbers regardless of the presence or absence of endogenous p53. However, overexpression of the NDRG1 failed to directly induce cell death and to alter susceptibility to apoptosis induced by tumor necrosis factor-/cycloheximide. These results indicate that NDRG1 is one of the direct mediators of induced p53 following polyamine depletion and that p53-dependent NDRG1 expression plays a critical role in the negative control of IEC proliferation.