Effects of exogenous oxidative stress on glutathione production by Saccharomyces cerevisiae mutant Y518 were researched by addition of exogenous hydrogen peroxide. The results showed that the addition of hydrogen peroxide inhibited the growth of Y518 cells, but promoted the synthesis of glutathione of Y518. The best addition time and amount of hydrogen peroxide were respectively 24 h and 0.6 mmol/L. The content of gluconate reached (13.69±0.28) mg/g, increased by about 14% compared to that without hydrogen peroxide. Exogenous oxidation stress on Y518 was produced by adding 0.6 mmol/L hydrogen peroxide at 24 h, which activated the transcription regulation factor YAP1 and SKN7 to control expressions of γ-glutamylcysteine synthetase, then that of glutathione synthetase encoded genes GSH I and GSH II, and finally enhanced the activities of γ-glutamylcysteine and glutathione synthetases to promote glutathione synthesis in Y518. Therefore, the addition of exogenous hydrogen peroxide could produce exogenous oxidation stress on Y518 and further promote Y518 to synthesize glutathione.