INTRODUCTION: Heat stress severely impairs plant growth and crop productivity. WRINKLED1 (WRI1), an AP2/EREBP-class transcription factor in Arabidopsis thaliana, orchestrates carbon partitioning between glycolysis and fatty acid biosynthesis, playing pivotal roles in development and stress adaptation. Elucidating its molecular function under high-temperature stress is critical for improving thermotolerance in crops. RATIONALE: While WRI1’s metabolic regulatory function has been elucidated, its role in heat response remains unexplored. To decipher the molecular mechanism of WRI1-mediated thermotolerance, we integrated genetic approaches (wild type, wri1-4, and WRI1-OE (overexpression)) with qRT-PCR and phenotyping under controlled heat stress. RESULTS: In this study, histochemical GUS staining of pWRI1::GUS transgenic lines demonstrated constitutive WRI1 expression throughout Arabidopsis seedlings, with significantly enhanced transcription in cotyledons under heat stress (HS) (P <0.05). Prolonged HS induced gradual transcriptional attenuation, though the transcription levels remained elevated than under the optimal temperature (22°C). qRT-PCR confirmed thermo-responsive WRI1 upregulation (peak:1 h HS, 3-fold induction), followed by threshold-dependent decline, indicating acute early-phase responsiveness. Endogenous immunoassays revealed reduced WRI1 protein accumulation under HS, suggesting HS-impaired protein stability or post-translational regulatory mechanisms. Thermotolerance phenotyping of WT, wri1-4, and WRI1-OE lines showed superior HS survival rate in WRI1-OE, with acquired thermotolerance exceeding basal thermotolerance across genotypes, confirming WRI1-mediated positive thermoregulation. The survival rate of WRI1-OE seedlings reached approximately 75%–85%, whereas that of wild type and complementary lines was less than 10%. We found that WRI1-OE reduced reactive oxygen species (ROS) accumulation, where direct transcriptional regulation of HSF/ HSP genes (e.g., HSFA2, HSP101) was excluded by qRT-PCR. Nevertheless, differential gene expression across genotypes indicated WRI1’s auxiliary role in thermotolerance via ROS scavenging and indirect proteostasis maintenance. CONCLUSION: This study proved WRI1 as a master regulator of thermotolerance, functioning through synergistic activation of chaperone networks ( HSFA2-HSPs) and ROS scavenging. The discovery of its cross-pathway coordination mechanism provides novel insights into plant thermal adaptation, and positions WRI1 as a potentially useful target for breeding climate-resilient crops.