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To investigate the effects of pH stress coupled with cysteine addition on glutathione (GSH) production in the treatment of high cell density culture of Candida utilis.We have previously observed that most Candida utilis cells remained viable after being subjected to pH at 1·2 for 3 h and that some intracellular GSH leaked into the medium. A cysteine addition strategy was applied in fed-batch production of GSH. A single cysteine addition resulted in higher GSH yield than two separate additions without pH stress. An increase in intracellular GSH content triggered inhibition of γ-glutamylcysteine synthetase (γ-GCS). A strategy that combines cysteine addition with low-pH stress was developed to relieve the inhibition of γ-GCS.Without pH stress, single shot and double shot cysteine addition yielded a total GSH of 1423 and 1325 mg l−1. In comparison, a low-pH stress counterpart resulted in a total GSH of 1542 and 1730 mg l−1, respectively. With low-pH stress, we observed GSH secretion into the medium at 673 and 558 mg l−1 and an increase in the γ-GCS activity by 1·2- and 1·5-fold, respectively. The specific GSH production yield increased from 1·76% to 1·91% (w/w) for single shot, and 1·64% to 2·14% for double shots.Low-pH shift was applied to alleviate the feedback inhibition of intracellular GSH on γ-GCS activity by secreting GSH into the medium. This strategy is coupled with cysteine addition to enhance GSH production in Candida utilis.