The effect of additives including yeast extract, precursor amino acids and ATP on the production of glutathione (GSH) by recombinant E. coli was investigated. The addition of precursor amino acids (glutamate, cysteine and glycine) and ATP improved intracellular GSH accumulation. When 2.0 g/l ATP and 9 mmol/l precursor amino acids were added at the beginning and after 12 h of fermentation, respectively, cell concentration and intracellular GSH content was 24% and 1.4 times higher than that of culture without additions. Growth inhibition was observed while the initial glucose concentration was above 20 g/l. Based on the analysis of the batch culture process, different fed-batch cultures in jar fermenters were carried out in which three kind of simple feeding strategies were applied. An exponential feeding strategy increased cell density, productivity and biomass yield. Using this strategy, cell density and total GSH quantity in broth reached 80 g(DCW)/l and 880 mg/l at 25 h without pure oxygen, respectively.