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Heat stress is a severe environmental factor that significantly reduces plant growth and delays development. Heat stress factors (HSFs) are a class of transcription factors that are synthesized rapidly in response to elevations in temperature and are responsible for the transcription of many heat stress-responsive genes including those encoding heat shock proteins (HSPs). There are 21 HSFs in Arabidopsis, and recent studies have established that the HSFA1 family members are master regulators for the remaining HSFs. However, very little is known about upstream molecular factors that control the expression of HSFA1 genes and other HSF genes under heat stress. Through a forward genetic analysis, we identified RCF3, a K homology (KH) domain-containing nuclear-localized putative RNA-binding protein. RCF3 is a negative regulator of most HSFs, including HSFA1a, HSFA1b, and HSFA1d. In contrast, RCF3 positively controls the expression of HSFA1e, HSFA3, HSFA9, HSFB3, and DREB2C. Consistently with the overall increased accumulation of heat-responsive genes, the rcf3 mutant plants are more tolerant than the wild-type to heat stress. Together, our results suggest that a KH domain-containing putative RNA-binding protein RCF3 is an important upstream regulator for heat stress-responsive gene expression and thermotolerance in Arabidopsis.