Intrahepatic metastasis is one of the most important prognostic factors for patients with hepatocellular carcinoma (HCC). Cell motility mediated by Rho- and p160 Rho-associated coiledcoil forming protein kinase (p160ROCK) signaling pathways has recently been shown to play a critical role in intrahepatic metastasis in human HCC. Furthermore, the stable introduction of dominant-negative p160ROCK into Li7 cells resulted in a reduced metastatic rate in mice with severe combined immunodeficiency (SCID). To investigate whether the specific p160ROCK inhibitor, Y-27632, could also inhibit intrahepatic metastasis, the effect of Y-27632 on the cell motility and intrahepatic metastasis of Li7 was investigated. Y-27632 markedly blocked actin reorganization and motility of Li7 cells mediated by lysophosphatidic acid (LPA). Y-27632 was administered continuously into the peritoneal cavity using a micro-osmotic pump, together with orthotopic implantation of Li7 cells into the liver of SCID mice. Phosphate-buffered saline (PBS) alone was administered as the control. The incidence of mice with metastatic nodules decreased in the Y-27632-treated group. The primary tumor volume at the site of injection was smaller in the Y-27632-treated group compared with the control group, but the difference was not statistically significant. Histologically, control tumors showed infiltrative growth into the sinusoidal area at the tumor boundary, whereas Y-27632-treated tumors showed expansive growth and low invasiveness. These findings confirm the importance of the Rho/p160ROCK signaling pathway in intrahepatic metastasis of human HCC, and indicate that Y-27632 may be useful for the prevention of intrahepatic metastasis of human HCC.