Abstract: Objective: To elucidate the cause for ceased cAMP formation during the later stage of fermentation with auxiliary energy substance added and develop coping strategy to further improve cAMP production. Methods: cAMP fermentations with sodium citrate and sodium pyruvate respective addition were conducted in a 7 L fermenter and corresponding fermentation kinetics, cells viability, energy metabolism and ROS levels were analyzed. Finally, a fermentation process with glutathione and auxiliary energy substance coupling added was proposed and conducted for improving cAMP contents. Results: The cAMP formation and cells growth rates in energy substance added conditions were higher than those of control obviously at the initial stage and dropped down at 30 h rapidly to low levels lower than control. The cells viability, ATP/AMP and ETC activities also dropped down at 36 h rapidly to low levels with larger decline than control. The analysis results indicated that reactive oxygen species (ROS) and malonaldehyde contents increased rapidly after 36 h due to the addition of auxiliary energy substance, which were higher than those of control obviously (P<0.01), whereas NADPH/NADP⁺ dropped sharply at the same time holding at lower levels than control. Finally, with the proposed fermentation process coupling glutathione and auxiliary energy substance added, cell and cAMP concentrations were improved obviously due to higher cell viability. cAMP concentrations in citrate coupling glutathione added batch and pyruvate coupling glutathione added batch achieved 4.05 and 4.32 g/L, respectively, with increments of 15.2% and 22.7% when compared with those of only GSH added batch, and with increments of 21.9% and 30.1% when compared with control(without auxiliary energy substance and glutathione addition). Conclusion: Auxiliary energy substance aroused plentiful ROS formation due to the intensive electron leakage from electron transport chain which caused cell injury and ceased cAMP synthesis. The fermentation process with glutathione and auxiliary energy substance coupling added relieved cell damage from ROS and further enhanced cell viability and cAMP fermentation production.