We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic β-cells. In an extracellular solution with 5.5 mm glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca²⁺ channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic β-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from −90 to −50 mV induced membrane currents mostly through ATP-sensitive K⁺ channels (K_ATP). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca²⁺ chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through K_ATP with 0.5 mm EGTA, but not with 10 mm EGTA, in the pipette solution. Intracellular application of GDPβS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca²⁺ concentration inside the membrane from 10 nm–10 μm decreased K_ATP activity only in the presence of ATP. The affinity of ATP to K_ATP became 4.5 times higher with the higher concentration of Ca²⁺. These results suggest that Ca²⁺ from ACh receptor signaling modulates the sensitivity of K_ATP to ATP. A positive-feedback mechanism of intracellular Ca²⁺-dependent Ca²⁺ influx was also demonstrated.