ATP-sensitive K⁺ (K_ATP) channels are activated by several vasodilating hormones and neurotransmitters through the protein kinase A (PKA) pathway. Here we show that phosphorylation at Ser1387 of the SUR2B subunit is critical for the channel activation. Experiments were performed in HEK293 cells expressing the cloned Kir6.1/SUR2B channel. In whole-cell patch, the Kir6.1/SUR2B channel activity was stimulated by isoproterenol via activation of ₂ receptors. This effect was blocked in the presence of inhibitors for adenylyl cyclase or PKA. Similar channel activation was seen by exposing inside-out patches to the catalytic subunit of PKA. Since none of the previously suggested PKA phosphorylation sites accounted for the channel activation, we performed systematic mutational analysis on Kir6.1 and SUR2B. Two serine residues (Ser1351, Ser1387) located in the NBD2 of SUR2B were critical for the channel activation. In vitro phosphorylation experiments showed that Ser1387 but not Ser1351 was phosphorylated by PKA. The PKA-dependent activation of cell-endogenous K_ATP channels was observed in acutely dissociated mesenteric smooth myocytes and isolated mesenteric artery rings where activation of these channels contributed drastically to the isoproterenol-induced vasodilation. Taken together, these results indicate that the Kir6.1/SUR2B channel is a target of ₂ receptors, and the channel activation relies on PKA phosphorylation of SUR2B at Ser1387.