Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N‐terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N‐terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co‐purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. J. Cell. Physiol. 231: 2682–2689, 2016. © 2016 Wiley Periodicals, Inc. Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. Examination of the N‐terminal extension of the isoform A suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co‐purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function.