The co-culture system of the fermentation process of vitamin C can be regarded as an artificial microbial ecosystem (AME). To extend our understanding of this AME, an investigation of the relationship between strains, substrate and product was carried out in this study. The results showed that both Ketogulonicigenium vulgare and 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) can inhibit the growth of the helper strain, while the helper strain promoted the growth of K. vulgare and 2-KLG production. Moreover, L-sorbose is not only a substrate for 2-KLG production in the AME, but also a promoter of K. vulgare and an inhibitor of the helper strain. In the earlier stage of fermentation, the inhibition of L-sorbose on the helper strain's growth is a key factor for ensuring an efficient fermentation. In the condition of adding the extra helper strain (OD: 0.57, ratio of inoculation: 2%), the yields of 2-KLG is increased by 9% in the 14% L-sorbose medium. To the best of our knowledge, this is the first report about the inhibition of substrate in the AME of 2-KLG production.