The effects of pH, broth volume, initial sugar concentration, ratio of carbon to nitrogen and phosphorus, and the glucose feeding method on GSH production in a shaking flask were investigated. The results showed that the proper pH and broth content for GSH production were 6.0 and 60 ml broth per 500 ml flask, respectively. The initial glucose concentration distinctly affected the GSH production; the intracellular GSH content of yeast would decrease when the initial glucose concentration was beyond 12 g/L. A glucose feeding strategy with the purpose of controlling the specific growth rate at an expected value was developed and applied to a 12 hour fermentation with the total glucose concentration 26.2 g/L; the final cell concentration (DCW) and the intracellular GSH content could reach 8.78 g/L and 13.6 mg/g, respectively, while the total GSH in the broth was 119.4 mg/L and the yield of cell to glucose was 0.335 g/g.