Fundamental to the metabolic sensor function of ATP-sensitive K+ (KATP) channels is the sulfonylurea receptor. This ATP-binding cassette protein, which contains nucleotide binding domains (NBD1 and NBD2) with conserved Walker motifs, regulates the ATP sensitivity of the pore-forming Kir6.2 subunit. Although NBD2 hydrolyzes ATP, a property essential in KATP channel gating, the role of NBD1, which has limited catalytic activity, if at all, remains less understood. Here, we provide functional evidence that cooperative interaction, rather than the independent contribution of each NBD, is critical for KATP channel regulation. Gating of cardiac KATP channels by distinct conformations in the NBD2 ATPase cycle, induced by γ-phosphate analogs, was disrupted by point mutation not only of the Walker motif in NBD2 but also in NBD1. Cooling membrane patches to decelerate the intrinsic ATPase activity counteracted ATP-induced KATP channel inhibition, an effect that mimicked stabilization of the MgADP-bound posthydrolytic state at NBD2 by the ω-phosphate analog orthovanadate. Temperature-induced channel activation was abolished by mutations that either prevent stabilization of MgADP at NBD2 or ATP at NBD1. These findings provide a paradigm of KATP channel gating based on integration of both NBDs into a functional unit within the multimeric channel complex.