Ammonia detoxification and gluconeogenesis are major hepatic functions mutually connected through amino acid metabolism. The liver is rich in glutamate dehydrogenase (GDH) that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate and ammonia, thus bridging amino acid-to-glucose pathways. Here we generated inducible liver-specific GDH-knockout mice (HepGlud1-/- ) to explore the role of hepatic GDH on metabolic homeostasis. Investigation of nitrogen metabolism revealed altered ammonia homeostasis in HepGlud1-/- mice characterized by increased circulating ammonia associated with reduced detoxification process into urea. The abrogation of hepatic GDH also modified energy homeostasis. In the fasting state, HepGlud1-/- mice could barely produce glucose in response to alanine due to impaired liver gluconeogenesis. Compared with control mice, lipid consumption in HepGlud1-/- mice was favored over carbohydrates as a compensatory energy fuel. The changes in energy partitioning induced by the lack of liver GDH modified the circadian rhythm of food intake. Overall, this study demonstrates the central role of hepatic GDH as a major regulator for the maintenance of ammonia and whole-body energy homeostasis.