Islets are highly vascularized for prompt insulin secretion. Although angiopoietin-1 (Ang1) is a well-known angiogenic factor, its role in glucose homeostasis remains largely unknown. The objective of this study was to investigate whether and how Ang1 contributes to glucose homeostasis in response to metabolic challenge. We used inducible systemic Ang1 knockout (Ang1sys-/-) and β-cell-specific Ang1 knockout (Ang1β-cell-/-) mice fed a high-fat diet for 24 weeks. Although the degree of insulin sensitivity did not differ between Ang1sys-/- and Ang1sys+/+ mice, serum insulin levels were lower in Ang1sys-/- mice, resulting in significant glucose intolerance. Similar results were observed in Ang1β-cell-/- mice, suggesting a critical role of β-cell-derived Ang1 in glucose homeostasis. There were no differences in β-cell area or vasculature density, but glucose-stimulated insulin secretion was significantly decreased, and PDX-1 expression and GLUT2 localization were altered in Ang1β-cell-/- compared with Ang1β-cell+/+ mice. These effects were associated with less pericyte coverage, disorganized endothelial cell ultrastructure, and enhanced infiltration of inflammatory cells and upregulation of adhesion molecules in the islets of Ang1β-cell-/- mice. In conclusion, β-cell-derived Ang1 regulates insulin secretion and glucose homeostasis by stabilizing the blood vessels in the islet and may be a novel therapeutic target for diabetes treatment in the future.