In this study, we tested for the temporal occurrence of photosynthetic acclimation to elevated [CO2] in the flag leaf of two important cereal crops, rice and wheat. In order to characterize the temporal onset of acclimation and the basis for any observed decline in photosynthetic rate, we characterized net photosynthesis, gs, gm, Ci/Ca, Ci/Cc, Vcmax, Jmax, cell wall thickness, content of Rubisco, cytochrome (Cyt) f, N, chlorophyll and carbohydrate, mRNA expression for rbcL and petA, activity for Rubisco, sucrose phosphate synthase (SPS) and sucrose synthase (SS) at full flag expansion, mid-anthesis and the late grain-filling stage. No acclimation was observed for either crop at full flag leaf expansion. However, at the mid-anthesis stage, photosynthetic acclimation in rice was associated with RuBP carboxylation and regeneration limitations, while wheat only had the carboxylation limitation. By grain maturation, the decline of Rubisco content and activity had contributed to RuBP carboxylation limitation of photosynthesis in both crops at elevated [CO2]; however, the sharp decrease of Rubisco enzyme activity played a more important role in wheat. Although an increase in non-structural carbohydrates did occur during these later stages, it was not consistently associated with changes in SPS and SS or photosynthetic acclimation. Rather, over time elevated [CO2] appeared to enhance the rate of N degradation and senescence so that by late-grain fill, photosynthetic acclimation to elevated [CO2] in the flag leaf of either species was complete. These data suggest that the basis for photosynthetic acclimation with elevated [CO2] may be more closely associated with enhanced rates of senescence, and, as a consequence, may be temporally dynamic, with significant species variation.