We previously identified osteopontin (OPN) as a promoter and thus a potential therapeutic target for hepatocellular carcinoma (HCC) metastasis. The serine protease thrombin interacts with OPN and can modify its biological activity. To explore the role of thrombin alone or in conjunction with OPN in HCC, we studied the correlation of thrombin levels to HCC prognosis in patients with various OPN levels, and evaluated the effects of OPN fragments generated by thrombin cleavage on proliferation and adhesion of HCC cells. We found that the thrombin level was strongly associated with the metastatic potential of HCC cell lines, and that thrombin was remarkably overexpressed in HCC tissue compared with adjacent nontumor tissue. In addition, HCC tissue from patients with recurrent disease displayed much higher thrombin levels, particularly in those with elevated OPN levels. Only HCCs with elevated OPN levels had a significant correlation between high thrombin levels and overall survival (OS; P < 0.01), or time to recurrence (TTR; P < 0.0001) of HCC. Multivariate analysis revealed that thrombin was an independent prognostic indicator. In vitro assays demonstrated that thrombin promotes the proliferation and adhesion of OPN+ HCC cells. Furthermore, thrombin activated the focal adhesion kinase (FAK) pathway of OPN+ HCC cells, which was blocked by the inhibition of integrin β1. Conclusion: Thrombin plays an important role in OPN-mediated aggressive phenotype of HCC through activation of integrin β1-FAK signaling, and is an independent poor prognostic factor for HCC. Thus, thrombin may be a potential therapeutic target to inhibit HCC metastasis in OPN+ patients (HEPATOLOGY 2010.)