Deregulation of the ERK5 pathway has been shown to be associated with various oncogenic processes, including metastatic potential of prostate cancer cells, and growth and chemoresistance of breast cancer cells. The aim of this study was to understand the role of ERK5 in hepatocellular carcinoma (HCC). ERK5 was silenced by siRNA transfection. Cell proliferation was evaluated by MTT assay. Cell cycle progression was analyzed by flow cytometry analysis. HCC xenografts were obtained using Huh/ and athymic Nu/nu mice. The ERK5 inhibitor XMD8-92 was used at the dose of 50 mg/kg i.p., bid. The role of ERK5in vitrowas studied by ERK5 knockdown by siRNA and pharmacological inhibition using XMD8-92. Treatment of HepG2 or Huh-7 with EGF activated ERK5, and stimulated cell migration and invasion. These effects were significantly reduced in ERK5-silenced cells or following specific inhibition. Confocal microscopy immunofluorescence showed that ERK5 silencing or inhibition is associated with remodeling of the cytoskeleton and focal adhesions, in keeping with a less motile phenotype. Inhibition of ERK5 activity also caused growth arrest in HCC cells lines, affecting the G1/S transition. Also when cultured in conditions of hypoxia (3% O2) the motile and invasive phenotype was dependent on activation of ERK5. In human HCC specimens, ERK5 staining was abundantly localized in the nucleus of neoplastic cells, indicating the activated status of the molecule. To test the involvement of ERK5 in HCC growhin vivo,we treated nude mice xenografted with Huh-7 cells with the ERK5 inhibitor, XMD8-92. Preliminary results indicate that ERK5 inhibition reduces the volume of the xenografted tumors without apparent adverse effects. ERK5 appear therefore to be involved in several features of HCC suggesting that this kinase may be an appealing target for the treatment of HCC.