Gallbladder cancer (GBC) is a malignant tumor of the biliary tract. The main problem affecting the treatment of gallbladder cancer is late diagnosis and poor prognosis. EIF5A2 is one of two isoforms of the EIF5A family and is reported to be a new oncogenic protein in many human cancers. In this study, our results showed for the first time that EIF5A2 was overexpressed in GBC samples compared with non-tumor tissue. Overexpression of EIF5A2 was associated with lymph node metastasis, tumor differentiation, UICC (Union for International Cancer Control) staging, histological type, metastasis, and tumor size. Overexpression of EIF5A2 in gallbladder carcinoma tissues is also associated with poor prognosis in patients. The interference of EIF5A2 significantly inhibited the proliferation, cell cycle, migration and colony formation of GBC-SD cells in vitro. Our results suggest that EIF5A2 is a target oncogene and may be an important prognostic biomarker in the pathogenesis of gallbladder cancer.