Background: Serum amino acids, part of a pool of free amino acids, are influenced by metabolism and disease. Few reports exist describing their function and concentrations in serum. Our study aimed to determine age- and gender-related differences in serum amino acid concentrations in healthy Japanese patients, focusing on their inclusion in body proteins. Methods: Healthy patients with normal liver and kidney function were selected based on biochemical variables. Serum amino acid concentrations in 23–88-year-old patients (312 males, 252 females; 52.1±15.0 years) were evaluated to determine the normal values and to clarify the differences in concentration. The patients were divided into 11 groups according to their gender and age (in 5-year increments), and serum amino acid concentrations were determined as the normal values for each group. Results: Some amino acids increased or decreased with age. Values of leucine, threonine, methionine, histidine, glycine, serine and taurine decreased with age in males, whereas, those of threonine and serine decreased with age in females (p<0.05). In contrast, values of phenylalanine, aspartate, asparagine, glutamate, alanine, citrulline, ornithine, arginine and tyrosine increased with age in males, whereas, values of phenylalanine, lysine, asparagine, glutamate, glutamine, alanine, citrulline, ornithine, arginine, tyrosine and proline increased with age in females (p<0.05). Conclusions: We clarified basic trends of age- and gender-related amino acid concentrations in serum. In normal healthy Japanese people who ate normally and lived an independent life, there are significant age- and gender-related differences.