Background and Purpose: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure.Experimental Approach: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain.Key Results: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin.The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats.In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric Abbreviations: albVHH, albumin binding variable heavy chain domain of a heavy-chain antibody; CMC, chemistry, manufacturing, and controls; FcRn, neonatal Fc receptor; NHP, nonhuman primate; RXFP1, relaxin family peptide receptor 1; RXFP2, relaxin family peptide receptor 2; SPR, surface plasmon resonance.