Background: The effect of relaxin and spironolactone combined on myocardial fibrosis has not been reported. Thus, we investigated the effect of the combined therapy on isoprenalineinduced myocardial fibrosis and the mechanism. Methods: Rats were injected subcutaneously with isoprenaline to induce myocardial fibrosis and underwent subcutaneous injection with relaxin (2 μg·kg-1·d-1) and given a gavage of spironolactone (30 mg·kg-1·d-1) alone or combined for 14 days. In vitro, the endothelial-mesenchymal transition was induced with transforming growth factor β (TGF-β) in human umbilical vein endothelial cells (HUVECs) pretreated with relaxin, 200 ng/ml, and/or spironolactone, 1uM. Results: Relaxin and spironolactone used alone or combined improved cardiac function and decreased cardiac weight indices; reduced fibrous tissue proliferation; reduced levels of type I and III collagen; decreased the expression of α- smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1), and increased the expression of cluster of differentiation-31 (CD31) in rats with isoprenaline-induced myocardial fibrosis. In vitro, compared with TGF-β treatment, relaxin and spironolactone used alone or combined with TGF-β decreased cell mobility, α-SMA and vimentin levels but increased vascular endothelial cadherin (VE-cadherin) and endothelial CD31levels. Especially, combined therapy had more remarkable effect than relaxin and spironolactone used alone both in vitro and in vivo. Conclusion: Relaxin and spironolactone combined affected isoprenaline-induced myocardial fibrosis in rats that the mechanism might be inhibition of the cardiac endothelial- mesenchymal transition. [ABSTRACT FROM AUTHOR]