Background Previously, we have demonstrated that the transplantation of mitochondria into the myocardium significantly enhanced cardioprotection. Our studies established that mitochondria are internalized into cardiomyocytes following transplantation; however, the mechanism(s) modulating internalization of these organelles was unknown. Methods: Neonatal rat cardiomyocytes were cultured and pre-treated with either cytochalasin D to block actin polymerization, methyl-β-cyclodextrin to block caveola-dependent-clathrin dependent endocytosis, nocodazole to block tunneling nano tubes or 5-(N-Ethyl-N-isopropyl)amiloride to block macro-pinocytosis and then co-incubated with isolated mitochondria pre-labeled with a fluorescent marker (pHrodo). Mitochondrial internalization and ATP content was determined. The functional contribution of internalized mitochondria, was determined using HeLa p0 cells depleted of mtDNA co-incubated with mitochondria isolated from HeLa cells with intact mtDNA. Results: Internalization of mitochondria into cardiomyocytes was modulated by actin-dependent endocytosis. Internalization of HeLa cell mitochondria into HeLa p0 cells increased ATP content (12.74±4.6 vs 73.7±17.2 nmol ATP/ 103 cells; p<0.05) and oxygen consumption rate (0.55±0.21 vs. 2.32±0.94; p<0.05) and replaced depleted mtDNA. Conclusions These results provide a mechanism for the internalization of mitochondria within host cells and a basis for novel therapeutic interventions allowing for the rescue and replacement of damaged or impaired mitochondria.