Metastasis is a critical factor contributing to poor prognosis in cancer, but the underlying mechanisms of metastasis are still poorly understood. We established a highly metastatic cell subline (HOC313-LM) derived from an oral squamous cell carcinoma cell line (HOC313) for uncovering the mechanisms of metastasis, and identified deoxyhypusine synthase (DHPS) as a metastasis-associated gene within the specific amplification at 19p13.2-p13.13 in HOC313-LM. DHPS-mediated hypusine-modification of eukaryotic translation factor 5A facilitated the translation of RhoA, resulting in the activation of the RhoA signaling pathway and leading to not only increased cell motility, invasion and metastasis of cancer cells in vitro, but also increased tumor growth in vivo. Moreover, the use of N1-Guanyl-1,7-diaminoheptane, a DHPS inhibitor, resulted in a significant decrease in tumor formation in vivo. In patients with esophageal squamous cell carcinoma (ESCC), overexpression of DHPS in ESCC tumors was significantly associated with worse recurrence-free survival, and correlated with distant metastasis. The elucidation of these molecular mechanisms within the hypusine cascade suggests opportunities for novel therapeutic targets in SCC.