Glucagon secreted from the pancreatic alpha-cells is essential for regulation of blood glucose levels. However, glucagon may play an equally important role in the regulation of amino acid metabolism by promoting ureagenesis. We hypothesized that disruption of glucagon receptor signaling would lead to an increased plasma concentration of amino acids, which in a feedback manner stimulates the secretion of glucagon, eventually associated with compensatory proliferation of the pancreatic alpha-cells. To address this, we performed plasma profiling of glucagon receptor knockout (Gcgr^-/-) mice and wild-type (WT) littermates using liquid chromatography mass spectrometry (LC-MS)-based metabolomics, and tissue biopsies from the pancreas were analyzed for islet hormones and by histology. A principal component analysis of the plasma metabolome from Gcgr^-/- and WT littermates indicated amino acids as the primary metabolic component distinguishing the two groups of mice. Apart from their hyperaminoacidemia, Gcgr^-/- mice display hyperglucagonemia, increased pancreatic content of glucagon and somatostatin (but not insulin), and alpha-cell hyperplasia and hypertrophy compared to WT littermates. Incubating cultured α-TC1.9 cells with a mixture of amino acids (Vamin 1%) for 30 minutes and for up to 48 hours led to increased glucagon concentrations (~six-fold) in the media and cell proliferation (~two-fold), respectively. In anesthetized mice, a glucagon receptor specific antagonist (Novo Nordisk 25-2648, 100 mg/kg) reduced amino acid clearance. Our data supports the notion that glucagon secretion and hepatic amino acid metabolism are linked in a close feedback loop, which operates independently of normal variations in glucose metabolism.