FOXQ1 emerges as a master transcriptional regulator of brain endothelial metabolism, orchestrating mitochondrial function through dual control of calcium signaling and cristae organization. This study reveals that brain endothelial cells rely on oxidative phosphorylation rather than glycolysis alone, challenging the current metabolic paradigm and providing new mechanistic insights into cerebrovascular specialization with important implications for neurological diseases. Abstract The blood‐brain barrier (BBB) maintains brain homeostasis through specialized functions including tight junction formation and selective transport of brain endothelial cells (ECs). While ECs are generally thought to rely primarily on glycolysis for energy production, the transcriptional mechanisms underlying their metabolic specialization in the brain endothelium remain poorly understood, especially considering the brain's extraordinary energy demands. Through comparative transcriptomic analysis, it is demonstrated that brain endothelial cells are enriched for mitochondrial function genes, with forkhead box protein 1 (FOXQ1) being selectively expressed in cerebral vasculature. Conditional knockout of Foxq1 in endothelial cells results in severe mitochondrial dysfunction, including disrupted cristae morphology, reduced oxygen consumption, and impaired adenosine triphosphate (ATP) production. Mechanistically, FOXQ1 directly regulates two key pathways: calcium signaling through huntingtin‐associated protein (HAP1)‐mediated endoplasmic reticulum (ER)‐mitochondrial calcium transfer, and mitochondrial structural integrity via AarF domain‐containing protein kinase 1 (ADCK1)‐dependent cristae organization. These findings reveal that brain endothelial cells rely on oxidative phosphorylation rather than glycolysis alone, challenging the prevailing metabolic paradigm for endothelial cells. This work establishes FOXQ1 as an important regulator of brain endothelial metabolism and provides new insights into the molecular basis of cerebrovascular specialization, with implications for understanding vascular dysfunction in neurological diseases. FOXQ1 emerges as a master transcriptional regulator of brain endothelial metabolism, orchestrating mitochondrial function through dual control of calcium signaling and cristae organization. This study reveals that brain endothelial cells rely on oxidative phosphorylation rather than glycolysis alone, challenging the current metabolic paradigm and providing new mechanistic insights into cerebrovascular specialization with important implications for neurological diseases. Abstract The blood-brain barrier (BBB) maintains brain homeostasis through specialized functions including tight junction formation and selective transport of brain endothelial cells (ECs). While ECs are generally thought to rely primarily on glycolysis for energy production, the transcriptional mechanisms underlying their metabolic specialization in the brain endothelium remain poorly understood, especially considering the brain's extraordinary energy demands. Through comparative transcriptomic analysis, it is demonstrated that brain endothelial cells are enriched for mitochondrial function genes, with forkhead box protein 1 (FOXQ1) being selectively expressed in cerebral vasculature. Conditional knockout of Foxq1 in endothelial cells results in severe mitochondrial dysfunction, including disrupted cristae morphology, reduced oxygen consumption, and impaired adenosine triphosphate (ATP) production. Mechanistically, FOXQ1 directly regulates two key pathways: calcium signaling through huntingtin-associated protein (HAP1)-mediated endoplasmic reticulum (ER)-mitochondrial calcium transfer, and mitochondrial structural integrity via AarF domain-containing protein kinase 1 (ADCK1)-dependent cristae organization. These findings reveal that brain endothelial cells rely on oxidative phosphorylation rather than glycolysis alone, challenging the prevailing metabolic paradigm for endothelial cells. This work establishes FOXQ1 as an important regulator of brain endothelial metabolism and provides new insights into the molecular basis of cerebrovascular specialization, with implications for understanding vascular dysfunction in neurological diseases. Advanced Science, Volume 12, Issue 42, November 13, 2025.