Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O₂ constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O₂ alters the function of an O₂ sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H₂O₂), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K⁺ channels, Kv). O₂ constriction was evaluated in 26 human DAs (12 female, aged 9±2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O₂ cause similar constriction and K⁺ current inhibition (I_K). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O₂ and 4-AP constriction in DA rings and reduced O₂- and 4-AP–sensitive I_K in DASMCs. Remodeled DAMSCs are depolarized and express less O₂-sensitive channels (including Kv2.1, Kv1.5, Kv9.3, Kv4.3, and BK_Ca). Kv2.1 adenoviral gene-transfer significantly reverses ionic remodeling, partially restoring both the electrophysiological and tone responses to 4-AP and O₂. In fresh DASMCs, ETC inhibitors (rotenone and antimycin) mimic hypoxia, increasing I_K and reversing constriction to O₂, but not phenylephrine. O₂ increases, whereas hypoxia and ETC inhibitors decrease H₂O₂ production by altering mitochondrial membrane potential (ΔΨm). H₂O₂, like O₂, inhibits I_K and depolarizes DASMCs. We conclude that O₂ controls human DA tone by modulating the function of the mitochondrial ETC thereby varying ΔΨm and the production of H₂O₂, which regulates DASMC Kv channel activity and DA tone.