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 collagens I and III were reduced. The collagen I / III ratio was also reduced. The mRNA and protein expression levels of TGF- β1, TβR I , 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.