We performed a review of public microarray data that revealed a significant down-regulation of Rnd3 expression in hepatocellular carcinoma (HCC), as compared to nontumor liver. Rnd3/RhoE is an atypical RhoGTPase family member because it is always under its active GTP-bound conformation and not sensitive to classical regulators. Rnd3 down-regulation was validated by quantitative real-time polymerase chain reaction in 120 independent tumors. Moreover, Rnd3 down-expression was confirmed using immunohistochemistry on tumor sections and western blotting on human tumor and cell-line extracts. Rnd3 expression was significantly lower in invasive tumors with satellite nodules. Overexpression and silencing of Rnd3 in Hep3B cells led to decreased and increased three-dimensional cell motility, respectively. The short interfering RNA-mediated down-regulation of Rnd3 expression induced a loss of E-cadherin at cell-cell junctions that was linked to epithelial-mesenchymal transition through the up-regulation of the zinc finger E-box binding homeobox protein, ZEB2, and the down-regulation of miR-200b and miR-200c. Rnd3 knockdown mediated tumor hepatocyte invasion in a matrix-metalloproteinase-independent, and Rac1-dependent manner. CONCLUSION: Rnd3 down-regulation provides an invasive advantage to tumor hepatocytes, suggesting that RND3 might represent a metastasis suppressor gene in HCC.