We previously reported the in vitro and in vivo hepatoprotective and anti-fibrotic effects of PF2401-SF, a standardized fraction of Salvia miltiorrhiza, against acute and subacute liver injury. The aim of this study was to investigate the effect of PF2401-SF on liver fibrosis induced by thioacetamide (TAA), a chronic liver injury model (12 weeks) that closely resembles fibrosis and cirrhosis in humans. Hepatoprotective activity was indicated by low serum levels of the markers aspartate amino transferase and alanine amino transferase .In addition, compared to the TAA-group livers, the PF2401-SF-treated liver tissues showed no fibrous tissue deposition in the portal areas, hepatocyte morphology more closely resembling normal tissue morphology, and significantly reduced collagen deposition. Furthermore, downregulation of collagen 1(α) and tissue inhibitor of metalloproteinase (TIMP)1 protein and mRNA expression also supports PF2401-SF's anti-fibrotic effect. We also observed reduced expression of α-smooth muscle actin (α-SMA), an important marker of hepatic stellate cells (HSCs) activation. From these results, we conclude that PF2401-SF's anti-fibrotic mechanism in the TAA model involves reduced HSC activation, and may be mediated by downregulation of central markers of fibrosis, including collagen 1(α), TIMP1, and α-SMA.