Vascular ATP-sensitive K+ channels are activated by multiple vasodilating hormones and neurotransmitters via PKA. A critical PKA phosphorylation site (Ser1387) is found in the second nucleotide-binding domain (NBD2) of SUR2B subunit. To understand how phosphorylation at Ser1387 leads to changes in channel activity, we modeled the SUR2B using a newly crystallized ABC protein SAV1866. The model showed that the Ser1387 was located on the interface of NBD2 with TMD1 and physically interacted with Tyr506 in TMD1. A positively charged residue (Arg1462) in NBD2 was revealed in the close vicinity of Ser1387. Mutation of either these three residues abolished the PKA-dependent channel activation. Molecular dynamics simulations suggested that Ser1387, Tyr506 and Arg1462 formed a compact triad upon Ser1387 phosphorylation, leading to reshaping of the NBD2 interface and movements of NBD2 and TMD1. Restriction of the interdomain movements by engineering a disulfide bond between TMD1 and NBD2 prevented the channel activation in a redox-dependent manner. Thus, a channel-gating mechanism is suggested via enhancing the NBD-TMD coupling efficiency following Ser1387 phosphorylation, which is shared by multiple vasodilators.