© 2014 The Authors. Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals. Synopsis In vivo analysis reveals a synergistic role of mitochondrial fission protein Drp1 and Parkinson's disease-associated ligase parkin in the regulation of ubiquitination and degradation of mitochondria in the heart and brain. Mitochondria divide in cardiomyocytes. Drp1 deficiency causes mitochondrial dysfunction, lethal heart failure and neurodegeneration due to defects in mitophagy. Mitochondria enlarge and accumulate ubiquitinated outer membrane proteins and mitophagy adaptor protein p62 independently of parkin. Parkin is dispensable for mitochondrial respiration, heart function and neuronal survival in the presence of Drp1-regulated mitophagy. Simultaneous loss of Drp1 and parkin increases mitophagy defects. In vivo analysis reveals a synergistic role of mitochondrial fission protein Drp1 and Parkinson's disease-associated ligase parkin in the regulation of ubiquitination and degradation of mitochondria in the heart and brain.