Gas exchange, chlorophyll (Chl) fluorescence, photosynthetic pigments, and the expression of Rubisco and Rubisco activase in flag leaves of rice (Oryza sativa L.) from the heading to mature grain stage were investigated. The results showed that the photosynthetic capacity declined after full expansion of flag leaves. The decline of photosynthetic rate (Pn) in two cultivars during natural senescence was accompanied by a decrease in Chl content, as well as in the Rubisco and Rubisco activase contents. The Rubisco and Rubisco activase contents in Zhenong 966 decreased faster than that in Zhenong 952. The Pn diminished without a decrease in intercellular CO2 concentration during the early senescence of flag leaves, indicating that lower photosynthetic capacity is not caused by stomatal limitation but by reduced carboxylation efficiency. During the senescence of flag leaves, the correlation between the change in photosynthetic capacity and the variation in the abundance of Rubisco and Rubisco activase suggested that the decline in Pn of flag leaves could be attributed to the lower level of rbcS and rca transcripts. The (φ)PSⅡ and the electron transport rate appeared in the same rate as Pn. However, excitation pressure (1-qP) showed a different pattern and there was an inverse linear correlation between increased excitation pressure and the reduced Rubisco. Therefore, it is likely that the increased excitation pressure resulted from saturation of the electron transport chain, owing to a limitation of the reductant used by the Calvin cycle; in addition, the change in excitation pressure could further mediate the expression of the rbcS and rca genes, resulting in a fast reduction in Rubisco content.