Despite significant advances in therapies in past decades, the mortality rate of septic cardiomyopathy remains high. The aim of this study is to explore the therapeutic effects of combined treatment using melatonin and irisin in a mouse model of lipopolysaccharide (LPS)-mediated septic cardiomyopathy. Our data found that melatonin and irisin could further attenuate LPS-induced myocardial depression. Molecular investigation illustrated that melatonin and irisin cotreatment sustained cardiomyocyte viability and improved mitochondrial function under LPS stress. Pathway analysis demonstrated that macrophage-stimulating 1 (Mst1), which was significantly activated by LPS, was drastically inhibited by melatonin/irisin cotreatment. Mechanically, Mst1 activated c-Jun N-terminal kinase (JNK) pathway and the latter induced oxidative stress, adenosine triphosphate metabolism disorder, mitochondrial membrane potential reduction, and cardiomyocyte death activation. Melatonin and irisin cotreatment effectively inhibited the Mst1-JNK pathway and, thus, promoted cardiomyocyte survival and mitochondrial homeostasis. Interestingly, Mst1 overexpression abolished the beneficial effects of melatonin and irisin in vivo and in vitro. Altogether, our results confirmed that melatonin and irisin combination treatment could protect heart against sepsis-induced myocardial depression via modulating the Mst1-JNK pathways. © 2020 Wiley Periodicals, Inc.