AbstractThe regulatory role of eukaryotic translation initiation factor 5A1 (eIF5A1) in apoptosis was examined using HT‐29 and HeLa S3 cells. eIF5A is the only known protein to contain the unusual amino acid, hypusine, and eIF5A1 is one of two human eIF5A family members. Two observations indicated that eIF5A1 is involved in apoptosis. First, siRNA‐mediated suppression of eIF5A1 resulted in inhibition of apoptosis induced by various apoptotic stimuli, and second, adenovirus‐mediated over‐expression of eIF5A1 strongly induced apoptotic cell death. A mutant of eIF5A1 incapable of being hypusinated also induced apoptosis when over‐expressed indicating that unhypusinated eIF5A1 is the pro‐apoptotic form of the protein. Over‐expression of eIF5A1 or of the mutant resulted in loss of mitochondrial transmembrane potential, translocation of Bax to the mitochondria, release of cytochrome c, caspase activation, up‐regulation of p53, and up‐regulation of Bim, a pro‐apoptotic BH3‐only Bcl‐2 family protein. In addition, BimL and BimS, the pro‐apoptotic alternative spliced forms of Bim, were induced in response to over‐expression of eIF5A1. Thus eIF5A1 appears to induce apoptosis by activating the mitochondrial apoptotic pathway. Proteomic analyses indicated that, of 1,899 proteins detected, 131 showed significant changes in expression (P ≤ 0.05, expression ratio ≥1.5) within 72 h of eIF5A1 up‐regulation. Among these are proteins involved in translation and protein folding, transcription factors, proteins mediating proteolysis, and a variety of proteins known to be directly involved in apoptosis. These observations collectively indicate that unhypusinated eIF5A1 plays a central role in the regulation of apoptosis. J. Cell. Physiol. 223:798–809, 2010. © 2010 Wiley‐Liss, Inc.