Background: Sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (HCC) and thus sets the new standard for the first-line treatment of advanced HCC. However, it remains unresolved to predict the drug sensitivity in treating HCC with sorafenib. Pretreatment pERK level has been shown to be associated with favorable response to such therapy in a phase 2 preclinical study, indicating that pERK may be a potential biomarker for treatment of HCC with sorafenib. Methods: The effects of sorafenib and 5-Fluorouracil on cell proliferation were evaluated by cell viability assay in four types of HCC cell line (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6), with different metastatic potential and basal pERK expression. Levels of pERK expression were determined by immunocytochemical analysis and quantification, along with Western Blot analysis. Correlation analysis was carried out between the IC50 values of drugs and Mean Optical Density values of pERK. Results: The basal pERK levels increased stepwise in cell lines in accordance with their metastatic potential. Sorafenib inhibited ERK phosphorylation at a concentration between 5 and 20 M dose-dependently, while no changes were observed after 5-FU treatment. Correlation analysis between the IC50 values and MOD values of pERK revealed that the effects of sorafenib were significantly correlated with basal pERK levels (Spearman r=-0.8671, P=0.0003). On the other hand, the resistance to 5-FU were significantly associated with basal pERK expression in these HCC cell lines (Spearman r=0.7846, P=0.0009). Conclusions: In this vitro study, pERK was confirmed to be a useful biomarker predictive of sensitivity in treating HCC with sorafenib. The RAF/MEK/ERK pathway may be involved in invasion, metastasis and drug resistance to traditional chemotherapy in HCC. 