γ-Glutamylvalylglycine is known as a potent "kokumi" substance that increases the thickness, continuity, and mouthfulness of the taste of food. In this study, γ-glutamylvalylglycine was enzymatically synthesized from glutamine and valylglycine through the transpeptidation reaction of bacterial γ-glutamyltranspeptidase. The reaction conditions decided for the production of γglutamylvalylglycine are 20 mM glutamine, 100 mM valylglycine, 30 mU/mL γ-glutamyltranspeptidase, and 0.856 M (5%) NaCl at pH 8 and 37 °C, and 18.6 mM γ-glutamylvalylglycine was synthesized. Metal cations, Na + and Mg 2+ , improved the transpeptidation reaction of γ-glutamyltranspeptidase, and the production of γ-glutamylvalylglycine increased. Moreover, by feeding a suitable amount of glutamine every 5 h, the maximum production of γ-glutamylvalylglycine increased 1.7 times after 20 h of incubation.