Updated project metadata. Heat stress is one of the extreme weather disasters potently decreasing cereal plant production. Recent studies have demonstrated that protein degradation and rRNA homeostasis as well as transcription factors are involved in thermoresponse in plants. However, how 5-methylcytosine (m5C) RNA modification contribute to heat stress response in plant remains largely unknown. Herein, we identified OsNSUN2 as the mRNA m5C methyltransferase in rice. The osnsun2 mutants specifically displayed severe phenotypes of temperature- and light-dependent lesion mimic and heat stress hypersensitivity. Heat stress enhanced mRNAs m5C methylation involving photosynthesis and detoxification systems, such as β-OsLCY, OsHO2, OsPAL1, and OsGLYI4, in an OsNSUN2-dependent manner, which then increased the protein synthesis. Consistently, the photosystem of the osnsun2 mutants was vulnerable to high ambient temperature and failed to be repaired under tolerable heat stress. Furthermore, OsNSUN2 mutation reduced photosynthesis efficiency and accumulated excessive reactive oxygen species upon heat treatment. Our findings demonstrate an important mechanism of mRNA m5C-dependent heat acclimation in rice.