Polyamines are present in all living organisms, and their homeostasis is closely associated with human health and disease. Further, they are small aliphatic cations that exhibit multifunctional activities through interactions with acidic substances, thereby precluding our understanding of their molecular functions in biological processes. eIF5A1 and eIF5A2 share high amino acid sequence similarity, and hypusination, using spermidine, is essential for their functions. eIF5A1 is ubiquitously expressed in all tissues and is essential for normal cell growth, whereas eIF5A2 is often expressed in human cancer tissues; however, the functional differences between eIF5A1 and eIF5A2 remain unclear. Here, we found that eIF5A2 is regulated by polyamines at the translational level and that eIF5A2, rather than eIF5A1, is important for cancer cell growth. The translational initiation of eIF5A2 mRNA was negatively regulated by miR-6514-5p at the 5ʹ-UTR, and polyamines inhibited this miRNA function, facilitating eIF5A2 synthesis. A proteomic analysis of cells with either eIF5A1 or eIF5A2 silenced showed distinct profiles. In addition, polyamines upregulated the expression of four ribosomal proteins, particularly RPS27A, RPL36A, and RPL22L1, which are associated with cancer malignancy. Our findings reveal an important role for eIF5A2, regulated by polyamines and miR-6514-5p, in cancer cell proliferation, suggesting that the interaction between eIF5A2 and ribosomes, which regulate cancer progression, is a selective target for cancer treatment.