Osteopontin (OPN) is overexpressed in a variety of cancers including hepatocellular carcinoma (HCC), and is likely involved in the process of vasculogenic mimicry (VM) in some tumor cells. In this study, we explored whether OPN plays a role for VM in HCC. Metastatic MHCC97-H human HCC cells and non-metastatic Hep3B human HCC cells were compared for their abilities to establish VM. Three dimensional-culture assays showed that MHCC97-H cells but not Hep3B cells were able to form the chord-like structure that represents VM. Real-time RT-PCR arrays were used to detect gene expression profiles of the two HCC cell lines in three-dimensional culture. PCR array analyses revealed the increased expression of OPN in MHCC97-H cells forming VM compared with Hep3B cells. Small interfering RNA was employed to investigate whether OPN knockdown could influence VM, and the expression of matrix metalloproteinase (MMP)-2, MMP-9 and urokinase-type plasminogen activator (uPA) in MHCC97-H cells. OPN knockdown resulted in a significant decrease in the ability of MHCC97-H cells to form VM, which was accompanied by the down-regulation of MMP-2 and uPA expression. Furthermore, human HCC tissue samples were studied by immunohistochemistry to analyze the correlations between VM and the expression of OPN, MMP-2 and uPA. There existed significant positive correlations between VM and the expression of OPN, MMP-2 and uPA in HCC tissue samples. In conclusion, OPN is required for VM in HCC cells, and its action may be associated with activation of MMP-2 and uPA. OPN-targeted therapeutics may be useful for patients with advanced HCC.