With global warming and climate change, breeding crop plants tolerant to high-temperature stress is of immense significance. tRNA 2-thiolation is a highly conserved form of tRNA modification among living organisms. Here, we report the identification of SLG1 (Slender Guy 1), which encodes the cytosolic tRNA 2-thiolation protein 2 (RCTU2) in rice. SLG1 plays a key role in the response of rice plants to high-temperature stress at both seedling and reproductive stages. Dysfunction of SLG1 results in plants with thermosensitive phenotype, while overexpression of SLG1 enhances the tolerance of plants to high temperature. SLG1 is differentiated between the two Asian cultivated rice subspecies, indica and japonica, and the variations at both promoter and coding regions lead to an increased level of thiolated tRNA and enhanced thermotolerance of indica rice varieties. Our results demonstrate that the allelic differentiation of SLG1 confers indica rice to high-temperature tolerance, and tRNA thiolation pathway might be a potential target in the next generation rice breeding for the warming globe.