Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of a rice plants 9,311 (Oryza sativa L.) cDNA in rice plants zhonghua11 (Oryza sativa L.). The genetic engineering enabled the plants to accumulate SBPase in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO(2) assimilation of transgenic plants was significantly more tolerant to high temperature than that of wild-type plants. The analyses of chlorophyll fluorescence and the content and activation of SBPase indicated that the enhancement of photosynthesis to high temperature was not related to the function of photosystem II but to the content and activation of SBPase. Western blotting analyses showed that high temperature stress led to the association of SBPase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than that in transgenic plants. The results in this study suggested that under high temperature stress, SBPase maintained the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fraction and thus enhanced the tolerance of CO(2) assimilation to high temperature stress. The results suggested that overexpression of SBPase might be an effective method for enhancing high temperature tolerance of plants.