Abstract: Glutathione is a major peptide protecting cells against oxidative stress. To study the cellular processes affecting intracellular glutathione production, we screened Saccharomyces cerevisiae mutant collections and identified new eight yeast deletion mutants that produced more than 1.2-fold higher levels of intracellular glutathione: chc1, cst6, ddc1, def1, pep12, rts1, ubp6, and yih1. Furthermore, overexpression of the DEF1 and CYS4 genes led to a higher production of glutathione, similar to overexpression of GSH1. A multiplier effect on activation of glutathione synthesis was observed by a combination of overexpression of GSH1 and deletion of one of the eight genes. Metabolome analysis of the def1, pep12, and ubp6 deletion mutant, and DEF1-overexpressing strains showed that levels of intracellular methionine and oxidized glutathione were higher than in the control strains, suggesting that methionine biosynthesis was activated and the oxidative stress response was increased in these glutathione-overproductive strains. Moreover, overexpression of GSH1, CYS4, and DEF1 also increased glutathione production in Candida utilis. Taken together, these results will significantly contribute to more effective industrial production of glutathione using yeasts.