In this study, we investigated the significance of β₂-adrenergic receptor (β₂AR) in age-related impaired insulin secretion and glucose homeostasis. We characterized the metabolic phenotype of β₂AR-null C57Bl/6N mice (β₂AR^−/−) by performing in vivo and ex vivo experiments. In vitro assays in cultured INS-1E β-cells were carried out in order to clarify the mechanism by which β₂AR deficiency affects glucose metabolism. Adult β₂AR^−/− mice featured glucose intolerance, and pancreatic islets isolated from these animals displayed impaired glucose-induced insulin release, accompanied by reduced expression of peroxisome proliferator–activated receptor (PPAR)γ, pancreatic duodenal homeobox-1 (PDX-1), and GLUT2. Adenovirus-mediated gene transfer of human β₂AR rescued these defects. Consistent effects were evoked in vitro both upon β₂AR knockdown and pharmacologic treatment. Interestingly, with aging, wild-type (β₂AR^+/+) littermates developed impaired insulin secretion and glucose tolerance. Moreover, islets from 20-month-old β₂AR^+/+ mice exhibited reduced density of β₂AR compared with those from younger animals, paralleled by decreased levels of PPARγ, PDX-1, and GLUT2. Overexpression of β₂AR in aged mice rescued glucose intolerance and insulin release both in vivo and ex vivo, restoring PPARγ/PDX-1/GLUT2 levels. Our data indicate that reduced β₂AR expression contributes to the age-related decline of glucose tolerance in mice.