Our previous studies have shown that polyamines are required for normal intestinal mucosal growth and that decreased levels of polyamines inhibit intestinal epithelial cell (IEC) proliferation by stabilizing p53 and other growth-inhibiting proteins. Nucleophosmin (NPM) is a multifunctional protein that recently has been shown to regulate p53 activity. In the present study, we sought to determine whether polyamine depletion increases NPM modulating the stability and transcriptional activity of p53 in a normal IEC-6 intestinal epithelial cell line. Depletion of cellular polyamines by alpha-difluoromethylornithine, the specific inhibitor of polyamine biosynthesis, stimulated expression of the NPM gene and induced nuclear translocation of NPM protein. Polyamine depletion stimulated NPM expression primarily by increasing NPM gene transcription and its mRNA stability, and it induced NPM nuclear translocation through activation of phosphorylation of mitogen-activated protein kinase kinase. Increased NPM interacted with p53 and formed a NPM/p53 complex in polyamine-deficient cells. Inhibition of NPM expression by small interfering RNA targeting NPM (siNPM) not only destabilized p53 as indicated by a decrease in its protein half-life but also prevented the increased p53-dependent transactivation as shown by suppression of the p21 promoter activity. Decreased expression of NPM by siNPM also promoted cell growth in polyamine-deficient cells. These results indicate that 1) polyamine depletion increases expression of the NPM gene and enhances NPM nuclear translocation and 2) increased NPM interacts with and stabilizes p53, leading to inhibition of IEC-6 cell proliferation.