S1FA-like proteins are a class of transcription factors that play significant roles in plant growth and development as well as in tolerance to abiotic stress. In this study, two S1FA-like family genes were identified in rice (Oryza sativa L.), and their biological functions in the response to heat stress were investigated. Bioinformatics analysis indicated that OsS1FA40 (LOC_Os04g33440) and OsS1FA20 (LOC_Os04g33420) genes have simple structures and high sequence homology. Expression pattern analysis revealed that OsS1FA40 is expressed in all tissues examined, whereas OsS1FA20 is predominantly expressed in the endosperm. Double mutant material of OsS1FA40 and OsS1FA20 in japonica rice Nipponbare (NIP) were obtained by using CRISPR-Cas9. Thermotolerance of Oss1fas mutants seedlings under heat treatment (42?°C, RH?>?90%, 48?h), the Oss1fas mutants exhibited a more thermotolerant phenotype compared to the wild type plants, with relatively less disruption to leaf cell morphology and structure. After recovery at 28?°C for one week, the mortality rate of the Oss1fas mutants was significantly lower than that of the WT plants. The analysis of physiological and biochemical related indicators showed that after heat treatment, the contents of chlorophyll A, chlorophyll B, total chlorophyll, soluble sugar and maximum photosynthetic efficiency in the Oss1fas mutants were significantly higher than those in the WT plants, indicating that heat stress activated the antioxidant mechanism and osmotic stress response of the Oss1fas mutants, and also affected the photosynthetic system. Furthermore, phenotypic analysis during plant development showed that the Oss1fas mutants displayed narrow and short leaves, as well as reduced root length, indicating potential involvement of these genes in leaf morphogenesis and plant growth regulation. In conclusion, OsS1FA in rice negatively regulate thermotolerance. This study provide a theoretical basis for deciphering the function of OsS1FA in regulating the response to high-temperature stress and for molecular breeding. This research holds significant implications and genetic improvement value for enhancing plant thermotolerance.