Endothelial cells (ECs) have a low mitochondrial density, around ~2–6% of cell volume, compared to cardiac cells (~32%). Consequently, ATP is generated mainly by the glycolytic pathway. The role of the mitochondrial network in ECs gains relevance as a signaling hub that modulates a wide range of endothelial functions, e.g., regulation of cell migration, proliferation, and angiogenesis, through the action of second messengers such as Ca2+ and reactive oxygen species (ROS). However, under stress conditions, such as lipotoxicity, the homeostasis of Ca2+ and ROS is disrupted due to an imbalance in the antioxidant system, leading to an oxidative stress state. In addition, the K+-channels are inhibited, causing membrane depolarization and contributing to the disruption of ionic homeostasis. This increases free Ca2+ in the cytosol and mitochondria, resulting in the opening of mitochondrial permeability transition (MPTP) and mitochondrial dysfunction. MPTP allows the escape of mitochondrial components, such as cytochrome c, ions, and even mitochondrial DNA (mtDNA) to the cytosol. Cytosolic mtDNA migrates to the endosome to interact with TLR-9 to activate the inflammation pathway. Then mtDNA is secreted by exosomes to propagate the inflammation signal to other cells. Endothelial dysfunction in obesity, metabolic syndrome, type 2 diabetes, diabesity, and cardiometabolic disease is promoted by lipotoxicity, increased oxidative stress, and inflammation. The relationship between these mechanisms likely involves the interaction of mtDNA and other mitochondrial components (e.g., HSP60) with pro-inflammatory receptors, e.g., TLR-9.