Liver fibrosis is characterized by excessive accumulation of extracellular matrix components in the liver parenchyma that distorts the normal architecture and hepatic function. Progressive fibrosis could end in the advanced stage known as cirrhosis, resulting in the need to resort to liver transplantation. Amniotic membrane (AM) has emerged as an innovative therapeutic approach for chronic liver diseases due to its anti-inflammatory, antiscarring, and wound-healing effects. We have recently shown that AM can be used as a patch on the liver surface at the same time of fibrosis induction, resulting in significantly reduced progression and severity of biliary fibrosis. Here we investigated the effects of human AM on the established rat model of liver fibrosis, induced by the bile duct ligation (BDL). We also explored the effect of AM on the expression of transforming growth factor-1 (TGF-1), the main profibrogenic factor in hepatic fibrosis, and the proinflammatory cytokines, tumor necrosis factor- (TNF-), interleukin-6 (IL-6), and anti-inflammatory cytokine IL-10. Two weeks after BDL, the liver was covered with a fragment of AM or left untreated. Six weeks later, the fibrosis was first assessed by the semiquantitative Knodell and the METAVIR scoring systems and, thereafter, by CellProfiler digital image analysis to quantify the area occupied by collagen deposition, ductular reactions (DRs), activated myofibroblasts, and TGF-1. The hepatic cytokines were determined by ELISA. AM-treated rats showed a significantly lower score compared to the control BDL rats (2.50.9 vs. 3.50.3, respectively; p0.05). The collagen deposition, DRs, number of activated myofibroblasts, and TGF-1 were all reduced to about 50% of levels observed in untreated BDL rats. These findings suggest that AM, when applied as a patch onto the liver surface, is useful for treating well-established cholestatic fibrosis, and the mechanism was partly by means of downregulating the profibrotic factor TGF-1 and IL-6.