Vasodilation in response to low oxygen (O) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O supply to tissues according to demand. However, how blood vessels react to O deficiency is not well understood. A common belief is that arterial myocytes are O-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Cachannels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O modulation of Ca channels and hypoxic vasodilation. Mitochondria function as O sensors and effectors that signal myocyte Ca channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.