Glutathione (GSH), an important tripeptide compound, is widely used as a therapeutic and in the food and cosmetic industries. To improve its production yield, we added the antibiotic nystatin to a batch fermentation of Saccharomyces cerevisiae, at different concentrations and at various times. Based on the results that nystatin can effectively stimulate GSH accumulation but at the same time inhibits cell growth, a three-point addition strategy (0.05 mg/L at 8 h, 0.25 mg/L at 16 h, and 0.5 mg/L at 20 h) was developed to maximize GSH production. As a result, a maximum yield of 237.8 mg/L was obtained, which was by 50.6% higher than without the addition of nystatin. When combining this strategy with cysteine addition, the GSH yield increased to 278.9 mg/L. Subsequently, the γ-glutamylcysteine synthetase (γ-GCS) activity and K+ concentration were analyzed to investigate the possible mechanism involved in the increased production. It was found that the nystatin-induced increase in the GSH yield was associated with a higher γ-GCS activity and K+ concentration.