The microbial co-culture system composing of Ketogulonicigenium vulgare and Bacillus cereus was widely adopted in industry for the production of 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. We found serial subcultivation of the co-culture could enhance the yield of 2-KGA by 16% in comparison to that of the ancestral co-culture. To elucidate the evolutionary dynamics and interaction mechanisms of the two microbes, we performed iTRAQ-based quantitative proteomic analyses of the pure cultures of K. vulgare, B. cereus and their co-culture during serial subcultivation. Hierarchy cluster analyses of the proteomic data showed that the expression level of a number of crucial proteins associated with sorbose conversion and oligopeptide transport was significantly enhanced by the experimental evolution. In particular, the expression level of sorbose/sorbosone dehydrogenase was enhanced in the evolved K. vulgare, while the expression level of InhA and the transport efficiency of oligopeptides were increased in the evolved B. cereus. The decreased sporulating protein expression and increased peptide transporter expression observed in evolved B. cereus, together with the increased amino acids synthesis in evolved K. vulgare suggested that serial subcultivation result in enhanced synergistic cooperation between K. vulgare and B. cereus, enabling an increased production of 2-KGA.