SUMMARY Heat stress triggers the accumulation of triacylglycerols in Arabidopsis leaves, which increases basal thermotolerance. However, how triacylglycerol synthesis is linked to thermotolerance remains unclear and the mechanisms involved remain to be elucidated. It has been shown that triacylglycerol and starch degradation are required to provide energy for stomatal opening induced by blue light at dawn. To investigate whether triacylglycerol turnover is involved in heat‐induced stomatal opening during the day, we performed feeding experiments with labeled fatty acids. Heat stress strongly induced both triacylglycerol synthesis and degradation to channel fatty acids destined for peroxisomal ß‐oxidation through the triacylglycerol pool. Analysis of mutants defective in triacylglycerol synthesis or peroxisomal fatty acid uptake revealed that triacylglycerol turnover and fatty acid catabolism are required for heat‐induced stomatal opening in illuminated leaves. We show that triacylglycerol turnover is continuous (1.2 mol% per min) in illuminated leaves even at 22°C. The ß‐oxidation of triacylglycerol‐derived fatty acids generates C2 carbon units that are channeled into the tricarboxylic acid pathway in the light. In addition, carbohydrate catabolism is required to provide oxaloacetate as an acceptor for peroxisomal acetyl‐CoA and maintain the tricarboxylic acid pathway for energy and amino acid production during the day.