The “bihormonal hypothesis”, published by Unger and Orci almost 50 years ago, proposes that Type 2 Diabetes (T2D) pathophysiology is a combination of insufficient insulin production by β-cells and elevated glucagon release by α-cells. Consistent with this hypothesis, several studies have illustrated that, in addition to an inappropriately low insulin secretory response, plasma glucagon levels in T2D patients remain elevated in response to high blood glucose levels. Despite decades of research, our understanding of how glucagon secretion is regulated remains ill-defined. To address this knowledge gap, my thesis work consists of two complementary studies investigating the role of the glycolytic enzyme glucokinase (GCK) and its opposing enzyme glucose-6-phosphatase (G6PC2) in the α-cell-intrinsic regulation of glucagon secretion. In our first study, we evaluated the effect of an α-cell-specific glucokinase activating mutation (α-mutGCK) on glucagon levels in mice, both under normal conditions and diabetogenic conditions of high-fat diet (HFD) feeding. We found that α-mutGCK mice present lower glucagon levels than controls in vivo, and demonstrate enhanced glucose-suppression of glucagon secretion (GSGS) ex vivo, independent of alterations in insulin levels and secretion, islet hormone content, or islet composition. Furthermore, α-mutGCK mice maintained on HFD displayed improvements in glucagonemia compared to control mice that developed the expected obesity, glucose intolerance, and hyperglucagonemia. Overall, these findings provide evidence that glucose sensing via glucokinase is a key determinant of glucagon secretion within mouse α-cells. In our second study, we evaluated the effect of α-cell-specific G6PC2 ablation (α-G6PC2KO) on glucagon release. Similar to the α-mutGCK mouse model, we found that α-G6PC2KO mice present lower glucagon levels than controls in vivo, as well as enhanced GSGS in isolated pancreatic islets, independent of alterations in insulin levels and secretion, islet hormone content, or islet composition. Notably, we also observed a greater suppression of glucagon from α-G6PC2KO mice in response to phloridzin treatment, suggesting that changes in α-cell-intrinsic glucose sensing, rather than alterations in α-cell insulin sensitivity, underlie our observed phenotype. Collectively, our data indicate that G6PC2 regulates glucagon release. Taken together, these studies contribute to our fundamental understanding of the cell-autonomous regulation of glucagon secretion via glucokinase and G6PC2.