Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca ²⁺ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca ²⁺ ] _i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca ²⁺ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca ²⁺ ] _i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca ²⁺ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca ²⁺ ] _i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca ²⁺ ] _i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca ²⁺ release from the acidic stores and further amplified by Ca ²⁺ -induced Ca ²⁺ release from the sarco/endoplasmic reticulum.