Central to fibrogenesis and the scarring of organs is the activation of fibroblasts into matrix-secreting myofibroblasts. We demonstrate that Galectin-3 expression is up-regulated in established human fibrotic liver disease and is temporally and spatially related to the induction and resolution of experimental hepatic fibrosis. Disruption of the Galectin-3 gene blocks myofibroblast activation and procollagen (I) expression in vitro and in vivo, markedly attenuating liver fibrosis. Addition of exogenous recombinant Galectin-3 in vitro reversed this abnormality. The reduction in hepatic fibrosis observed in the Galectin-3^−/− mouse occurred despite equivalent liver injury and inflammation, and similar tissue expression of TGF-β. TGF-β failed to transactivate Galectin-3^−/− hepatic stellate cells, in contrast with WT hepatic stellate cells; however, TGF-β-stimulated Smad-2 and -3 activation was equivalent. These data suggest that Galectin-3 is required for TGF-β mediated myofibroblast activation and matrix production. Finally, in vivo siRNA knockdown of Galectin-3 inhibited myofibroblast activation after hepatic injury and may therefore provide an alternative therapeutic approach to the prevention and treatment of liver fibrosis.