The global temperature has been continuously increasing in recent decades due to the explosion of population, the development of industries with emission of greenhouse gas, and excessive deforestation. High temperature in summer occurs more frequently and keeps longer in many regions of all over the world, leading to adverse effects on crop growth and development. Rice (Oryza sativa L.) is one of the most important staple food crops in China. The decrease in grain yield due to high temperature stress during the growth period has become a serious problem in rice production, especially in the Yangtze-Huaihe River Valley. Although effects of high temperatures on grain yield and quality have been studied, the mechanism involved in the injury of high temperature to rice growth is not understood. As proteomic approach is a new technique in plant stress physiology in recent years, this study investigated whether and how high temperature stress regulates protein expression in rice leaves. Two indica rice cultivars differing in heat tolerance, Shuanggui 1 (heat-sensitive) and Huanghuazhan (heat-tolerant), were pot-grown at Yangzhou University farm, Yanghzou, Jiangsu Province, China, and treated with high temperature stress at the stages of seedlings (during the 4th leaf, 38℃(day)/30℃(night)), meiosis (from 14 days before heading to the initial of heading, 35℃/25.6℃), heading (0-10 d after heading, 33.4℃/20.9℃) and early grain filling (11-20 d after heading, 33.4℃/20.9℃). The temperatures for controls were 25℃/20℃ at the seedling stage, 32.7℃/25.6℃ at meiosis, 29.9℃/20.9℃ at the heading, and 29.9℃/20.9℃ at the early grain filling stage. The protein expression in the leaf was observed by using two-dimensional gel electrophoresis with immobilized pH 4-7 gradients as the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension. The results showed that the changes in protein expressions under high temperature stress exhibited four statuses: some proteins were induced, some proteins were up-regulated, some proteins were inhibited, and some proteins were down-regulated. Protein expressions varied with the treatment stages and the two cultivars. Among the four treatment stages, protein expressions induced by high temperature stress were the most at the early grain filling stage, followed by the seedling stage, the third at meiosis, and the least at the heading stage. Protein expressions were more in the heat-sensitive cultivar than in the heat-tolerant cultivar. The mass spectrosmetric analysis indicated that the differences in protein expressions were mainly those involved in photosynthesis and signal transduction, such as Os01g0791600, Os12g0291400, ankyrin-like protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (large subunit), Hypothetical protein OsI_22016, and Rubisco activase small isoform precursor. These proteins were inhibited or down-regulated in the heat-sensitive cultivar Shuanggui 1, but were induced or up-regulated in the heat-tolerant cultivar Huanghuazhan. The results suggest that proteins involved in photosynthesis and signal transduction play a major role in heat tolerance of rice. The possible regulatory mechanism of these proteins in rice leaves in response to high temperature was discussed.