The translation factor eIF5A is a protein containing the amino acid residue hypusine, which has never been found in other proteins. In human cells, an EIF5A1 transcript variant referred to as variant A is expected to encode the eIF5A1 isoform A, which has an additional N-terminal extension with respect to the canonical isoform B. Here, we validated the isoform A in HeLa cells and showed that variant A is able to produce both eIF5A1 isoforms.