Aims Oxidative stress limited the growth of cells and 2‐keto‐l‐gulonic acid (2‐KGA) production in vitamin C (Vc) fermentation system. The study aims to investigate the antioxidant effect of glutathione on promoting 2‐KGA in Vc fermentation system using Ketogulonicigenium vulgare 25B‐1 and Bacillus endophyticus ST‐1 as the co‐culturing microbes. Methods and Results The activities of antioxidant‐related enzymes and qPCR were used to study the antioxidant effect of glutathione addition in Vc fermentation system. The addition of GSH and GSH/GSSG increased 2‐KGA production and decreased fermentation time, and the highest 2‐KGA production increased by 40·63% and the lowest fermentation time shortened to 60 h when the addition of optimal concentration ratio of GSH/GSSG was 50 : 1. Moreover, the increased production of 2‐KGA was accompanied by up‐regulated the activities of total antioxidant capacity (T‐AOC), total superoxide dismutase (T‐SOD), catalase (CAT) and over‐expressed oxidative stress‐related genes sod, gst, gr, zwf, gp, which resulted in scavenging reactive oxygen species to reduce oxidative stress in Vc fermentation system. Conclusions Glutathione showed a significant effect on increasing 2‐KGA production and decreasing fermentation time in Vc fermentation system. GSH/GSSG could maintain a dynamic balance with two forms of glutathione and the optimal concentration ratio of GSH/GSSG was 50 : 1. Significance and Impact of the Study Glutathione is proved to be effective to relieve oxidative stress. The promotion effects of GSSG and GSH on 2‐KGA production could help to further explore the optimization of co‐culture fermentation process for Vc industrial production.