The present study investigated rice leaf proteome in response to heat stress. Rice seedlings were subjected to a temperature of 42°C and samples were collected 12 and 24 h after treatment. Increased relative ion leakage and lipid peroxidation suggested that oxidative stress frequently was generated in rice leaves exposed to high temperature. 2‐DE, coupled with MS, was used to investigate and identify heat‐responsive proteins in rice leaves. In order to identify the low‐abundant proteins in leaves, samples were prefractionated by 15% PEG. The PEG supernatant and the pellet fraction samples were separated by 2‐DE, and visualized by silver or CBB staining. Approximately 1000 protein spots were reproducibly detected on each gel, wherein 73 protein spots were differentially expressed at least at one time point. Of these differentially expressed proteins, a total of 34 and 39 protein spots were found in the PEG supernatant and pellet fractions, respectively. Using MALDI‐TOF MS, a total of 48 proteins were identified. These proteins were categorized into classes related to heat shock proteins, energy and metabolism, redox homeostasis, and regulatory proteins. The results of the present study show that a group of low molecular small heat shock proteins (sHSPs) were newly induced by heat stress. Among these sHSPs, a low molecular weight mitochondrial (Mt) sHSP was validated further by Western blot analysis. Furthermore, four differentially accumulated proteins that correspond to antioxidant enzymes were analyzed at the mRNA level, which confirmed the differential gene expression levels, and revealed that transcription levels were not completely concomitant with translation. The identification of some novel proteins in the heat stress response provides new insights that can lead to a better understanding of the molecular basis of heat‐sensitivity in plants.