The semisynthetic plant alkaloid Halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSC). Results were compared to its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose-dependently at submicromolar concentrations. 200nM of HAL up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10-50-fold, resulting in a 2-3-fold increase of interstitial collagenase activity. Procollagen alpha1(I) and MMP-2 transcript levels were suppressed 2-3 fold, while expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NFkB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFkB dose-dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and –13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, while tissue inhibitor of metalloproteinase-1 (TIMP-1) was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration, and upregulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFkB. The remarkable induction of MMP-3 and -13 make HAL a promising agent for antifibrotic combination therapies.