Heat-shock transcription factors (HSFs) are crucial for regulating plant responses to heat and various stresses, as well as for maintaining normal cellular functions and plant development. HSFA9 and HSFA2 are two of the Arabidopsis class A HSFs and their expressions are dramatically induced in response to heat shock (HS) stress among all 21 Arabidopsis HSFs. However, the detailed biological roles of their cooperation have not been fully characterized. In this study, we employed an integrated approach that combined bioinformatics, molecular genetics and computational analysis to identify and validate the molecular mechanism that controls seed longevity and thermotolerance in Arabidopsis. The acquisition of tolerance to deterioration was accompanied by a significant transcriptional switch that involved the induction of primary metabolism, reactive oxygen species and unfolded protein response, as well as the regulation of genes involved in response to dehydration, heat and hypoxia. In addition, the cis-regulatory motif analysis in normal stored and controlled deterioration treatment (CDT) seeds confirmed the CDT-repressed genes with heat-shock element (HSE) in their promoters. Using a yeast two-hybrid and molecular dynamic interaction assay, it is shown that HSFA9 acted as a potential regulator that can interact with HSFA2. Moreover, the knock-out mutants of both HSFA9 and HSFA2 displayed a significant reduction in seed longevity. These novel findings link HSF transcription factors with seed deterioration tolerance and longevity.