Significance Vascular K ATP channels formed by the potassium channel Kir6.1 and its regulatory protein SUR2B maintain blood pressure in the physiological range. Overactivity of the channel due to genetic mutations in either Kir6.1 or SUR2B causes severe cardiovascular pathologies known as Cantú syndrome. The cryogenic electron microscopy structures of the vascular K ATP channel reported here show multiple, dynamically related conformations of the regulatory subunit SUR2B. Molecular dynamics simulations reveal the negatively charged ED-domain in SUR2B, a stretch of 15 glutamate (E) and aspartate (D) residues not previously resolved, play a key MgADP-dependent role in mediating interactions at the interface between the SUR2B and Kir6.1 subunits. Our findings provide a mechanistic understanding of how channel activity is regulated by intracellular MgADP.