Patients with type 2 diabetes (T2D) and patients with non-alcoholic fatty liver disease (NAFLD) frequently exhibit elevated plasma concentrations of glucagon (hyperglucagonemia). Hyperglucagonemia and alpha-cell hyperplasia may result from elevated levels of plasma amino acids when glucagon's action on hepatic amino acid metabolism is disrupted. We therefore measured plasma levels of glucagon and individual amino acids in patients with and without biopsy-verified NAFLD and with and without type T2D. Fasting levels of amino acids and glucagon in plasma were measured, using validated ELISAs and high-performance liquid chromatography, in obese, middle-aged individuals with I) normal glucose tolerance (NGT) and NAFLD, II) T2D and NAFLD, III) T2D without liver disease, and IV) NGT and no liver disease. Elevated levels of total amino acids were observed in participants with NAFLD and NGT compared to NGT controls (1,310±235 µM vs. 937±281 µM, p=0.03) and in T2D and NAFLD compared to T2D without liver disease (1,354±329 µM vs. 511±235 µM, p<0.0001). Particularly amino acids with known glucagonotropic effects (e.g. glutamine) were increased. Plasma levels of total amino acids correlated to plasma levels of glucagon also when adjusting for body mass index (BMI), glycated haemoglobin (HbA1c), and cholesterol levels (β=0.013±0.007, p=0.024). Elevated plasma levels of total amino acids associate with hyperglucagonemia in NAFLD patients independently of glycemic control, BMI or cholesterol - supporting the potential importance of a 'liver-alpha-cell axis' in which glucagon regulates hepatic amino acid metabolism. Fasting hyperglucagonemia as seen in T2D may therefore represent impaired hepatic glucagon action with increasing amino acids levels.