Myofibroblasts are specialized contractile cells that participate in tissue fibrosis and remodeling, including idiopathic pulmonary fibrosis (IPF). Mechanotransduction, a process by which mechanical stimuli are converted into biochemical signals, regulates myofibroblast differentiation. Relaxin is a peptide hormone that mediates antifibrotic effects through regulation of collagen synthesis and turnover. In this study, we demonstrate enhanced myofibroblast contraction in bleomycin-induced lung fibrosis in mice and in fibroblastic foci of human subjects with IPF, using phosphorylation of the regulatory myosin light chain (MLC₂₀) as a biomarker of in vivo cellular contractility. Compared with wild-type mice, relaxin knockout mice express higher lung levels of phospho-MLC₂₀ and develop more severe bleomycin-induced lung fibrosis. Exogenous relaxin inhibits MLC₂₀ phosphorylation and bleomycin-induced lung fibrosis in both relaxin knockout and wild-type mice. Ex vivo studies of IPF lung myofibroblasts demonstrate decreases in MLC₂₀ phosphorylation and reduced contractility in response to relaxin. Characterization of the signaling pathway reveals that relaxin regulates MLC₂₀ dephosphorylation and lung myofibroblast contraction by inactivating RhoA/Rho-associated protein kinase through a nitric oxide/cGMP/protein kinase G–dependent mechanism. These studies identify a novel antifibrotic role of relaxin involving the inhibition of the contractile phenotype of lung myofibroblasts and suggest that targeting myofibroblast contractility with relaxin-like peptides may be of therapeutic benefit in the treatment of fibrotic lung disease.