As one of the most important crops, rice provides the major food for more than half of the world population. However, its production is limited by many environmental factors, among which high temperature stress (HS) frequently occurs during anthesis and reduces its spikelet fertility. To explore the mechanism of HS tolerance in rice, we conducted a comparative proteomics analysis on the anthers between HS resistant and sensitive cultivars under different levels of high temperature. Under the same HS treatment, the resistant cultivar showed much higher spikelet fertility than the sensitive cultivar. Proteomic data showed that HS lead to the degradation of ribosomal proteins in the sensitive cultivar but not in the resistant one, which might result in the injury of protein biosynthetic machinery. In contrast, HS induced the increase of sHSP, -expansins and lipid transfer proteins in the resistant cultivar, which might contribute to its ability to tolerate HS. The results provide some new insights into the mechanism of rice HS response.