Tumor suppressor p53 plays a pivotal role in orchestrating mitochondrial remodeling by regulating their content, fusion/fission processes, and intracellular signaling molecules that are associated with mitophagy and apoptosis pathways. In order to determine a molecular mechanism underlying flow-mediated mitochondrial remodeling in endothelial cells, we examined, herein, the role of p53 on mitochondrial adaptations to physiological flow and its relevance to vascular function using endothelial cell-specific p53 deficient mice. We observed no changes in aerobic capacity, basal blood pressure, or endothelial mitochondrial phenotypes in the endothelial p53 mull animals. However, after 7 weeks of voluntary wheel running exercise, blood pressure reduction and endothelial mitochondrial remodeling (biogenesis, elongation, and mtDNA replication) were substantially blunted in endothelial p53 null animals compared to the wild-type, subjected to angiotensin II-induced hypertension. In addition, endothelial mtDNA lesions were significantly reduced following voluntary running exercise in wild-type mice, but not in the endothelial p53 null mice. Moreover, in vitro studies demonstrated that unidirectional laminar flow exposure significantly increased key putative regulators for mitochondrial remodeling and reduced mitochondrial reactive oxygen species generation and mtDNA damage in a p53-dependent manner. Mechanistically, unidirectional laminar flow instigated translocalization of p53 into the mitochondrial matrix where it binds to mitochondrial transcription factor A, TFAM, resulting in improving mtDNA integrity. Taken together, our findings suggest that p53 plays an integral role in mitochondrial remodeling under physiological flow condition and the flow-induced p53-TFAM axis may be a novel molecular intersection for enhancing mitochondrial homeostasis in endothelial cells.