Calorie restriction increases longevity of mammals and yeasts but this mechanism remains unclear. In this study, the role of glutathione on lifespan extension induced by calorie restriction was investigated by using a Saccharomyces cerevisiae strain deficient in glutathione synthesis (gsh1). We observed an increase in chronological lifespan of calorie-restricted gsh1 mutant cells, compared to WT (wild type) strain, which was associated with a reduction in the levels of oxidative stress biomarkers. The gsh1 strain showed an increase in cell yield under calorie restriction that was associated with a higher pyruvate kinase activity and a reduction in oxygen consumption and aconitase activity. This indicates that the respiratory metabolism is decreased in gsh1 mutant cells. The lifespan extension of gsh1 mutant cells did not represent an advantage at long term, since old cells of gsh1 strain showed a higher frequency of petite mutants. In addition, aged WT cells outlast aged gsh1 mutant cells in direct competition assays in a fresh medium. These results suggest that glutathione is required for the beneficial effects of calorie restriction on cellular longevity.