Fibrosis (extracellular matrix (ECM) accumulation) is the final end-point in diabetic cardiomyopathy. The current study evaluated the therapeutic effects of the anti-fibrotic hormone relaxin in streptozotocin (STZ)-treated transgenic mRen-2 rats, which undergo pathological and functional features similar to human diabetes. Twelve-week old hyperglycaemic mRen-2 rats, normoglycaemic control rats, and animals treated with recombinant human gene-2 (H2) relaxin (H2-RLX) from weeks ten-twelve were assessed for various measures of left ventricular (LV) fibrosis, hemodynamics and function, while the mechanism of relaxin's actions was also determined. Hyperglycaemic mRen-2 rats had increased LV collagen concentration (fibrosis) and gelatinase activity (all p<0.05 vs controls), but equivalent levels of interstitial collagenase and TIMP-1 to that measured in control rats. The increased LV fibrosis associated with diabetic animals led to significant alterations in the E/A wave ratio and E-wave deceleration time (both p<0.05 vs controls) in the absence of blood pressure changes, reflective of myocardial stiffness and LV diastolic dysfunction. H2-RLX treatment of diabetic rats led to significant decreases in interstitial and total LV collagen deposition (both p<0.05 vs diabetic group), resulting in decreased myocardial stiffness and improved LV diastolic function, without affecting non-diabetic animals. The protective effects of H2-RLX in diabetic rats were associated with a reduction in mesenchymal cell differentiation and TIMP-1 expression in addition to a promotion of ECM-degrading MMP-13 (all p<0.05 vs diabetic group), but were independent of blood pressure regulation. These findings demonstrate that relaxin is an anti-fibrotic with rapid-occurring efficacy, and may represent a novel therapy for the treatment of diabetes.