We show that vitamin A (all-trans-retinol) (VA) is required both for the maintenance of pancreatic β-cell and α-cell mass and for glucose-stimulated insulin secretion in adult mice. Dietary VA deprivation (VAD) causes greatly decreased pancreatic VA levels, hyperglycemia, and reduced insulin secretion. Adult mice fed VAD diets display remodeling of the endocrine pancreas, marked β-cell apoptosis, shifts to smaller islet size distributions, decreased β-cell mass, increased α-cell mass, and hyperglucagonemia. Importantly, although we induced VAD in the entire animal, the pancreatic β-cells are exquisitely sensitive to VAD-associated apoptosis compared with other cell types in other organs. VAD causes major reductions in levels of the VA intracellular binding protein Crbp1 and the retinoic acid-metabolizing enzyme Cyp26a1 specifically in larger islets, suggesting the use of these proteins as biomarkers for early endocrine mass abnormalities. In the VAD mice, the reductions in pancreatic islet sizes and the associated aberrant endocrine functions, which show similarities to the phenotype in advanced type 2 diabetes, result from reductions in pancreatic VA signaling. Reintroduction of dietary VA to VAD mice restores pancreatic VA levels, glycemic control, normal islet size distributions, β-cell to α-cell ratios, endocrine hormone profiles, and RARβ2 and RARγ2 transcript levels. Restoration of β-cell mass by reintroducing VA to VAD mice does not involve increased β-cell proliferation or neogenesis. Pharmacologic modulation of pancreatic VA signaling should be explored for the preservation and/or restoration of pancreatic β-cell mass and function in individuals with diabetes mellitus.