High temperature causes huge yield loss in rice. Heat-shock factors (Hsfs) are key transcription factors regulating the expression of heat stress-responsive genes, but natural variation and functional characterization of Hsfs have been seldom reported. A significant heat response locus was detected by genome-wide association study (GWAS) using green leaf area as an indicative trait. A miniature inverted-repeat transposable element (MITE) in the promoter of a candidate gene HTG3 (heat-tolerance gene on chromosome 3) was found to be significantly associated with heat-induced expression of HTG3 and heat tolerance (HT). The MITE-absent variant has been selected in heat-prone rice-growing regions. HTG3a, an alternatively spliced isoform encoding a functional Hsf, positively regulated HT at both vegetative and reproductive stages based on the results of HTG3a over-expression and knockout rice lines. The HTG3-regulated genes were enriched for heat shock proteins and jasmonic acid signaling. Two heat-responsive JASMONATE ZIM-DOMAIN (JAZ) genes were confirmed to be directly up-regulated by HTG3a, and one of them, OsJAZ9, positively regulates HT. We conclude that HTG3 plays an important role in HT through the regulation of JAZs and other heat-responsive genes. The MITE-absent allele may be valuable for HT breeding in rice.