Relaxin is a naturally occurring peptide hormone that mediates systemic hemodynamic and renal adaptive changes during pregnancy and abrogates aberrant scar tissue formation (fibrosis) in diverse pathogeneses. However, its efficacy relative to renin–angiotensin system blockade, the most effective antifibrotic strategy currently available, is not known. We compared the individual versus combined antifibrotic effects of serelaxin (a recombinant form of human gene-2 relaxin) and the angiotensin-converting enzyme inhibitor enalapril, in preventative (started before injury) and therapeutic (treatment of established fibrosis) strategies, in a mouse model of isoprenaline-induced cardiac injury (at 17 days). Changes in systolic blood pressure, organ hypertrophy, and tissue remodeling/fibrosis were assessed. Pretreatment with serelaxin (0.5 mg/kg per day via subcutaneous administration) alone reduced cardiac fibrosis to a greater extent than enalapril (200 mg/L via drinking water; equivalent to 48 mg/kg per day) alone (P<0.05 versus enalapril alone). Additionally, the combined effects of serelaxin and enalapril reduced cardiac fibrosis by at least 2-fold compared with enalapril alone, when administered preventatively or therapeutically; by suppressing transforming growth factor-β1 expression and phosphorylation of Smad2 (an intracellular regulator of transforming growth factor-β1 activity; both P<0.05 versus enalapril alone) to a greater extent. The effects of serelaxin were independent of blood pressure, while enalapril lowered systolic blood pressure in the model studied. These findings suggest that serelaxin alone and in combination with an angiotensin-converting enzyme inhibitor more effectively ameliorates fibrosis than angiotensin-converting enzyme inhibition alone in the diseased heart, in a clinically relevant experimental scenario.