Hepatocellular carcinoma (HCC) carries a poor prognosis with no effective treatment available other than liver transplantation for selected patients. Vascular invasion of HCC is one of the most important negative predictor of survival. As the regulation of invasion of HCC cells is not well understood, our aim was to study the mechanisms by which galectin 3, a β-galactosidase-binding lectin mediates HCC cell migration. HCC was induced by N-diethylnitrosamine in wild-type and galectin 3(-/-) mice, and tumor formation, histology, and tumor cell invasion were assessed. The galectin 3(-/-) mice developed significantly smaller tumor burden with a less invasive phenotype than the wild-type animals. Galectin 3 was upregulated in the wild-type HCC tumor tissue, but not in the surrounding parenchyma. Galectin 3 expression in HCC was induced by NF-κB transactivation as determined by chromatin immunoprecipitation assays. In vitro studies assessed the pro-migratory effects of galectin 3. The migration of hepatoma cells was significantly decreased after transfection by the galectin 3 siRNA and also after using the Rho kinase inhibitor Y-27632. The reorganization of the actin cytoskeleton, RhoA GTPase activity and the phosphorylation of MLC2 (myosin light chain 2) were decreased in the galectin 3 siRNA-transfected cells. In addition, in vitro and in vivo evidence showed that galectin 3 deficiency reduced hepatoma cell proliferation and increased their apoptosis rate. In conclusion, galectin 3 is an important lectin that is induced in HCC cells, and promotes hepatoma cell motility and invasion by an autocrine pathway. Targeting galectin 3 therefore could be an important novel treatment strategy to halt disease progression.