AIM: To investigate the protective effect of osthole (Ost) on APAP-induced liver injury in mice and its molecular mechanism. METHODS: We established the APAP-induced liver injury model in mice, and Ost was used to intervene. The expression of AST, ALT, SOD, ROS, MDA, LDH, GSH-PX in mice plasma were detected by biochemical method. HE staining was used to observe the changes of liver tissue structure. Immunofluorescence assay was used to detect the expression of Tif1γ and Smad4 in liver tissue. The mRNA expression of IL-1β, IL-6, TNF-α, Smad4, and Tif1γ were detected by qRT-PCR. Western blot was applied to assess the protein expression of Smad2/3 and pSmad2/3 in liver tissue. RESULTS: Compared with the control group, the liver structure destruction and hepatocyte death was increased, ALT, AST, ROS, MDA and LDH were increased, while SOD and GSH-PX were decreased, and the mRNA expressions of IL-1β, IL-6 and TNF-α were increased in the model group. Compared with the model group, the Ost intervention group had improved liver structure and decreased liver cell death; decreased ALT, AST, ROS, MDA and LDH, increased SOD and GSH-PX, and decreased expression of IL-1β, IL-6 and TNF-α mRNA. Compared with the control group, liver tissues of model mice showed increased expression of pSmad2/3, Smad4 protein and Smad4 mRNA, and decreased Tif1γ protein and mRNA. Compared with the model group, the liver tissues of the Ost intervention group showed decreased expression of pSmad2/3, Smad4 protein and Smad4 mRNA, and increased expression of Tif1γ protein and mRNA. CONCLUSION: Ost can improve liver function, reduce oxidative stress and inflammatory reaction, and protect hepatocyte damage induced by APAP in mice, which may be related to the up-regulation of Tif1γ and inhibition of TGF-β1/Smad signaling pathway.