Mitochondria are essential organelles involved in the maintenance of cell growth and function, and have been investigated as therapeutic targets in various diseases. Recent studies have demonstrated that direct mitochondrial transfer can restore cellular functions of cells with inherited or acquired mitochondrial dysfunction. However, previous mitochondrial transfer methods are inefficient and time-consuming. Here, we developed a simple and easy mitochondrial transfer protocol using centrifugation, which can be applied to any cell type. By our simple centrifugation method, we found that the isolated mitochondria could be successfully transferred into target cells, including mitochondrial DNA-deleted Rho0 cells and dexamethasone-treated atrophic muscle cells. We found that mitochondrial transfer normalised ATP production, mitochondrial membrane potential, mitochondrial reactive oxygen species level, and the oxygen consumption rate of the target cells. Furthermore, delivery of intact mitochondria blocked the AMPK/FoxO3/Atrogene pathway underlying muscle atrophy in atrophic muscle cells. Taken together, this simple and rapid mitochondrial transfer method can be used to treat mitochondrial dysfunction-related diseases.