Chronic hepatitis/cirrhosis is the eighth leading cause of death in Taiwan. Excess accumulated extracellular matrix produced by activated hepatic stellate cells (HSCs) is the major cause of liver fibrosis. Ginsenoside Rb1, the most active compound purified from ginseng, has been considered to be hepatoprotective. This study investigated the effects of ginsenoside Rb1 (98.8% purity) on activation, proliferation, and profibrotic factors in rat HSC-T6 cells under H₂O₂ oxidative stress. Rat HSC-T6 cells were activated by 10 nM H₂O₂ and then incubated with different concentrations of ginsenoside Rb1 (5, 10, 20, 40, and 80 μg/mL) for 24 hours. Medium containing 0.08% dimethyl sulfoxide or 5 mM N-acetyl-l-cysteine was used as a negative or positive control, respectively. The results showed that ginsenoside Rb1 at 5-40 μg/mL significantly reduced α-smooth muscle actin levels and at 5-80 μg/mL inhibited cell proliferation in HSC-T6 cells after induction with H₂O₂ (P<.05). Collagen secreted by HSC-T6 cells was decreased by ginsenoside Rb1 at 5-80 μg/mL (P<.05). Protein expression of transforming growth factor-β1 (TGF-β1), matrix metalloproteinase (MMP)-2, and tissue inhibitor of metalloproteinase (TIMP)-1 was suppressed by ginsenoside Rb1 at 10-80 μg/mL (P<.05). In addition, mRNA expression of type I and III collagen, TGF-β1, and TIMP-1 was inhibited by ginsenoside Rb1 (10 and 80 μg/mL) (P<.05). Therefore, ginsenoside Rb1 exerted an antifibrotic effect on HSCs by inhibiting activation, proliferation, and expression of collagen, TGF-β1, MMP-2, and TIMP-1.