To explore the potential association between the diversity of endophytic microorganisms and modifications of grain quality in wheat exposed to multi-generational elevated CO2 concentration, the grain quality attributes and microbial diversity were tested after five generations successively grown in ambient CO2 concentration (F5_A, 400 μmol L−1) and elevated CO2 concentration (F5_E, 800 μmol L−1). Elevated CO2 concentration significantly increased the grain number and starch concentration, while decreased the grain protein concentration. Multi-generational exposure to elevated CO2 concentration also led to significant changes in grain amino acid concentration. In response to the elevated CO2 concentration, Pseudomonas, Rhodococcus, Ralstonia, and Klebsiella were the dominant bacterial genera, while Penicillium, Cutaneotrichosporon, Fusarium, Sarocladium, Acremonium and Aspergillus were the dominant fungal genera in wheat grain. A significantly positive correlation was found between Pseudomonas, Penicillium and ratio of starch to protein concentration, implying that the multi-generational CO2 elevation induced modifications in grain quality might be associated with the changes in grain microbial diversity. The results of this study suggest that the endophytic microbes may play an important role in modulating the grain nutritional quality in wheat under multi-generational e[CO2] exposure, through regulating starch and N metabolism and production of secondary metabolites.