ATP-sensitive potassium channels (KATP) are metabolic sensors that convert the intracellular ATP/ADP ratio to the excitability of cells. They are involved in many physiological processes and implicated in several human diseases. Here we present the cryo-EM structures of the pancreatic KATP channel in both the closed state and the pre-open state, resolved in the same sample. The nucleotides bind at the inhibitory sites of the Kir6.2 channel in the closed state but not in the pre-open state. Structural comparisons reveal the mechanism for ATP inhibition and Mg-ADP activation, two fundamental properties of KATP channels. Moreover, the structure also uncovers the activation mechanism of diazoxide-type KATP openers.