Several inorganic/organic N-containing substances were tested as nitrogen source for efficient glutathione production by C. utilis WSH 02-08. Although the strain could assimilate all the inorganic/organic nitrogen, urea and ammonium sulfate were found more favorable to cell growth and glutathione biosynthesis in a flask, respectively, and an optimal C/N ratio existed for each as 5.6 mol/mol and 8.3 mol/mol. A mixed nitrogen source of urea and ammonium under diverse C/N ratios could not boost glutathione fermentation despite the many mixed strategies that were introduced. Batch glutathione production in a stirred fermentor, using the sole or mixed nitrogen sources of urea and ammonium sulfate under their optimal C/N ratios, were conducted; urea was further proved to be the best nitrogen source for glutathione production. The reason was then quantitatively described by kinetic model, together with the distribution of flux for metabolites in metabolic network of glutathione biosynthesis by C. utilis WSH 02-08.