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Spring wheat (Triticum aestivum L. cv. Triso) was grown in a free-air CO2 enrichment (FACE) field experiment in order to gain information on CO2-induced effects on grain composition and quality at maturity. A proteome analysis was performed using two-dimensional gel electrophoresis (2-DE) and protein identification was done with mass spectrometry (MALDI-TOF MS). In elevated CO2 (526 μl l−1), an increase of 13.5% in grain yield was observed relative to 375 μl l−1 at a low level of significance (P = 0.528). Total grain protein concentration was decreased by 3.5% at a high level of statistical significance. Most importantly, a number of statistically significant changes within the grain proteome were observed, as the levels of 32 proteins were affected by elevated CO2: 16 proteins were up-regulated and 16 were down-regulated. Our experiment demonstrates that high-CO2 can markedly affect the proteome of mature wheat grain. The potential role of the proteins, changed in response to CO2 enrichment, is discussed as some may affect grain quality. For the task of selecting cultivars resistant to CO2-induced quality loss, we propose to consider the proteins affected by elevated CO2 identified in this work here.