The disruption of mitochondria homeostasis can impair the contractile function of cardiomyocytes, leading to cardiac dysfunction and an increased risk of heart failure. This study introduces a pioneering therapeutic strategy employing mitochondria derived from human umbilical cord mesenchymal stem cells (hu-MSC) (MSC-Mito) for heart failure treatment. Initially, we isolated MSC-Mito, confirming their functionality. Subsequently, we monitored the process of single mitochondria transplantation into recipient cells and observed a time-dependent uptake of mitochondria invivo. Evidence of human-specific mitochondrial DNA (mtDNA) in murine cardiomyocytes was observed after MSC-Mito transplantation. Employing a doxorubicin (DOX)-induced heart failure model, we demonstrated that MSC-Mito transplantation could safeguard cardiac function and avert cardiomyocyte apoptosis, indicating metabolic compatibility between hu-MSC-derived mitochondria and recipient mitochondria. Finally, through RNA sequencing and validation experiments, we discovered that MSC-Mito transplantation potentially exerted cardioprotection by reinstating ATP production and curtailing AMPKα-mTOR-mediated excessive autophagy.