Ghrelin reportedly serves as a physiological regulator of insulin release. This study aimed to explore signaling mechanisms for insulinostatic ghrelin action in islet β-cells, with special attention to heterotrimeric GTP-binding proteins and K⁺ channels. Plasma insulin and growth hormone (GH) concentrations in rats were measured by enzyme-linked immunosorbent assay (ELISA). Islets were isolated from rats, ghrelin-knockout (Ghr-KO) mice, and wild-type mice by collagenase digestion, and insulin release was determined by ELISA. In rat single β-cells, cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured by fura-2 microfluorometry, and membrane potentials and whole cell currents by patch-clamp technique. In rats, systemic ghrelin administration decreased plasma insulin concentrations, and this effect was blocked by treatment with pertussis toxin (PTX), whereas stimulation of GH release remained unaffected. In rat islets, ghrelin receptor antagonist increased and exogenous ghrelin suppressed glucose-induced insulin release in a PTX-sensitive manner. Glucose-induced insulin release from islets was greater in Ghr-KO than wild-type mice, and this enhanced secretion was blunted with PTX. Ghrelin PTX sensitively increased voltage-dependent K⁺ (Kv) currents without affecting ATP-sensitive K⁺ channels in rat β-cells. In the presence of Kv channel blockers, ghrelin failed to suppress insulin release. Ghrelin attenuated glucose-induced action potentials and [Ca²⁺]_i increases in β-cells. Suppressions of [Ca²⁺]_i increase and insulin release by ghrelin were blunted in β-cells treated with PTX and with antisense oligonucleotide specific for G-protein Gα_i2-subunit. Ghrelin attenuates glucose-induced insulin release via PTX-sensitive Gα_i2-mediated activation of Kv channels and suppression of [Ca²⁺]_i in β-cells, representing the unique signaling of ghrelin distinct from that for GH release.