The reliable truth that exceptional miRNAs contribute to gastric cancer (GC) development and progression had been testified by researchers. However, the roles of various miRNAs in GC remain to be determined. In present research, we firmly believed that the expression of miR-599 in GC cell lines and tissues was declined. Clinical analysis disclosed that poor prognostic features, which incorporated lymph node metastasis and advanced TNM stage, could be expressively influenced by down-regulation of miR-599. For 5-year predicted-survival of GC patients, miR-599 was also confirmed to be a specialty prognostic marker. Notably, overmuch expression of miR-599 restricted cell migration, invasion and EMT progress, while down-regulated miR-599 reversed the effect in vitro and in vivo. Through directly binding to the 3′-UTR, miR-599 could possess EIF5A2 efficiently. In clinical samples of GC, miR-599 was inversely correlated with EIF5A2, which was upregulated in GC. The influence of miR-599 on migration, invasion and EMT progress could be partially discarded through alternation of EIF5A2 expression. Conclusively, our results manifested that miR-599 possessed the function of tumor suppressor gene in regulating EMT and metastasizing GC via targeting EIF5A2. Therefore, miR-599 has the capacity to become a therapeutic target and prognostic marker for GC.