Previous studies indicated the grain yield and compositions of wheat (Triticum aestivum L.) were affected by long-term elevated atmospheric CO₂ concentration conditions. However, the roles of protein expression in wheat grain quality changes under multigenerational elevated atmospheric CO₂ concentration are still rarely known. This study explored that the changes of grain quality in wheat offspring induced by multigenerational elevated atmospheric CO₂ concentration exposure and analyzed the roles of the differently expressed proteins using 4D proteomics in regulating the wheat grain quality. The changes of grain protein accumulation, gluten index and dough development time indicated that the nutritional and end-use quality of wheat grains were directly affected by elevated atmospheric CO₂ concentration. This was mainly due to the changed expressions of α-amylase inhibitors, glutamine synthetase, glutamate dehydrogenase, formamidase and β-glucosidase, which regulated the starch accumulation and nitrogen metabolism in grains. This study elucidates the mechanisms underlying the effects of long-term elevated atmospheric CO₂ concentration on wheat grain quality.