Abstract: The present study was designed to elucidate whether the mechanism by which osthole decreases collagen Ⅰ/Ⅲ contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL^-1 of osthole for 24 h, the mRNA and protein expression levels of collagensⅠand Ⅲ were reduced. The collagen Ⅰ/Ⅲ ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRⅠ, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen Ⅰ and Ⅲ expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.