The eukaryotic initiation factor 5A (eIF5A) is an essential protein involved in translation elongation and cell proliferation. eIF5A undergoes several post-translational modifications including hypusination and acetylation. Hypusination is indispensable for the function of eIF5A. On the other hand, the precise function of acetylation remains unknown, but it may render the protein inactive since hypusination blocks acetylation. Here, we report that acetylation of eIF5A increases under hypoxia. During extended hypoxic periods an increase in the level of eIF5A acetylation correlated with a decrease in HIF-1 alpha, suggesting involvement of eIF5A activity in HIF-1 alpha expression under hypoxia. Indeed, suppression of eIF5A by siRNA oligo-mediated knockdown or treatment with GC7, a deoxyhypusine synthase inhibitor, led to significant reduction of HIF-1 alpha activity. Furthermore, knockdown of eIF5A or GC7 treatment reduced tumor spheroid formation with a concomitant decrease in HIF-1 alpha expression. Our results suggest that functional, hypusinated eIF5A is necessary for HIF-1 alpha expression during hypoxia and that eIF5A is an attractive target for cancer therapy. (C) 2016 Elsevier Inc. All rights reserved.