The past two decades have witnessed an evolving understanding of the mitochondrial genome's (mtDNA) role in basic biology and disease. From the recognition that mutations in mtDNA can be responsible for human disease to recent efforts showing that mtDNA mutations accumulate over time and may be responsible for some phenotypes of aging, the field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation. In this review, we critically discuss the past two decades of efforts and insights gained from cell and animal models of mtDNA mutation. We attempt to reconcile the varied and at times contradictory findings by highlighting the various methodologies employed and using human mtDNA disease as a guide to better understand cell and animal mtDNA models. We end with a discussion of scientific and therapeutic challenges and prospects for the future of mtDNA transfection and gene therapy.