Aspergillus oryzae is a filamentous fungus used for the manufacture of medicines and fermented foods. Glutathione is a tripeptide contained in variety of organisms and is involved in tolerance to various stresses. A. oryzae has a putative γ-glutamylcysteine synthetase gene (gshA) in its genome. To investigate the effect of the gene expression level of gshA on the amount of glutathione in A. oryzae, we generated a transformant where gshA expression was regulated by the thiamine-repressible thiA promoter. When the transformant was cultured on CzapekDox (CD) agar with added thiamine, the intracellular level of glutathione in the transformant decreased in a dose-dependent manner. Results of this study discovered that in oxidative stress conditions, repression of gshA gene expression with thiamine caused inhibition of growth of the transformant. Also, it was found that the gshA gene plays an important role in glutathione homeostasis in A. oryzae, and this gene may also be necessary for oxidative stress response.