Significance: Mitochondrial integrity is instrumental in protecting against damage associated with aging and a variety of chronic disease conditions. Mitochondrial silent information regulator 3 (Sirt3) plays pivotal roles in maintaining mitochondrial homeostasis by regulating different aspects of the organelle processes. Recent Advances: Mitochondria are highly dynamic organelles that constantly fuse and divide to maintain normal cell function, and perturbation in mitochondrial dynamics is responsible for mitochondrial dysfunction. Improved knowledge of mitochondrial physiology has disclosed the pleiotropic role of Sirt3 in mitochondria and shows how alterations in protein expression and/or activity may have an important impact on aging-associated organ dysfunction. Critical Issues: This review describes updated experimental evidence on the role of mitochondrial dysfunction during aging and renal diseases and highlights the emerging role of Sirt3 as a crucial regulator of mitochondrial dynamics. Future Directions: Strategies that activate Sirt3 may offer attractive therapies to achieve healthy longevity and preserve functional integrity of multiple organs. Antioxid. Redox Signal. 25, 185–199.