Glutamine is the most abundant amino acid in the human body and can be synthesized by almost all tissues by the glutamine synthetase (GS)-catalyzed amidation of glutamate. Hepatocytes have access to extracellular glutamine by the concentrative uptake via members of the sodium-dependent neutral amino acid transport systems N and A. Hepatic glutamine metabolism in connection with urea synthesis is importantly involved in systemic ammonia detoxication and pH regulation due to the unique regulatory properties of the liver-type glutaminase, the acinar compartimentation of urea and glutamine synthesis, and a cycling of glutamine between periportal and perivenous hepatocytes. Upregulation of GS expression in hepatocellular carcinoma is related to growth advantage and an enhanced metastatic potential. Glutamine is a potent activator of signal transduction. Recent progress concerns the understanding of glutamine-induced hepatocyte swelling and the downstream activation of integrins, Src, and MAP-kinases in the regulation of autophagic proteolysis, canalicular bile acid excretion, glycogen and fatty acid synthesis, insulin signaling, and protection from apoptosis. Most recently the first primary GS defect leading to inherited glutamine deficiency with fatal outcome was described in human. This review summarizes recent progress in the understanding of glutamine metabolism and signal transduction, which provides further rationale for the use of glutamine as a therapeutic tool.