The loss of inhibition of glucagon secretion exacerbates hyperglycemia in type 1 and 2 diabetes. However, the molecular mechanisms that regulate glucagon secretion in unaffected and diabetic states remain relatively unexplained. We present evidence supporting a new model of juxtacrine-mediated regulation of glucagon secretion where neighboring islet cells negatively regulate glucagon secretion through tonic stimulation of a-cell EphA receptors. Primarily through EphA4 receptors, this stimulation correlates with maintenance of a dense F-actin network. In islets, additional stimulation and inhibition of endogenous EphA forward signaling result in inhibition and enhancement, respectively, of glucagon secretion, accompanied by an increase and decrease, respectively, in α-cell F-actin density. Sorted a-cells lack endogenous stimulation of EphA forward signaling from neighboring cells, resulting in enhanced basal glucagon secretion as compared with islets and the elimination of glucose inhibition of glucagon secretion. Restoration of EphA forward signaling in sorted a-cells recapitulates both normal basal glucagon secretion and glucose inhibition of glucagon secretion. Additionally, a-cell-specific EphA4-/- mice exhibit abnormal glucagon dynamics, and EphA4-/- α-cells contain less dense F-actin networks than EphA4+/+ α-cells. This juxtacrine-mediated model provides insight into the functional and dysfunctional regulation of glucagon secretion and opens up new therapeutic strategies for the clinical management of diabetes.