Significance The slow delayed rectifier K ⁺ current ( I _Ks ) determines the length of each human heartbeat because it activates after myocytes are excited and depolarize. This sensitivity to voltage, as well as dynamic regulation by hormones and second messengers, underlies the essential role of I _Ks in determining the duration and rhythm of cardiac action potentials. Here, we demonstrate the unexpected mechanism that establishes the voltage-dependent operation of I _Ks channels: SUMOylation. When native I _Ks channels are resident in the plasma membranes of neonatal mouse ventricular myocytes, or human channels are reconstituted in CHO cells, each of the four KCNQ1 pore-forming subunits is subject to monoSUMOylation in a manner that depends on KCNE1 accessory subunits, leading to stepwise depolarizing shifts in the activation voltage.