Tribolium castaneum (Herbst) ranks as one of the most prevalent insects in food processing and storage facilities worldwide. Heat treatment has been revisited to disinfest food processing and storage facilities due to increasingly strict regulation on chemicals. The effect of acclimation of T. castaneum larvae to sublethal high temperatures of 36 and 42°C for 10 h on their heat adaptation was investigated, and transcript-level analysis combinating with real-time PCR (RT–qPCR) was applied for elucidating the heat adaptation mechanism of T. castaneum larvae. Short-term sublethal high temperature acclimation could greatly enhance the thermal adaptability in T. castaneum larvae. In total, 575, 875, and 1017 differentially expressed genes (DEGs) were, respectively, determined in comparisons between the 28 and 36°C treatments, the 28 and 42°C treatments, and the 36 and 42°C treatments. Fifty-three and 96 genes were commonly up- and down-regulated in both the 36 and 42°C treatments relative to 28°C, respectively. The results of RT-qPCR analysis further confirmed the RNA-seq analysis. The current results are in favor of enhancing the insecticidal effectiveness of extreme high temperature treatment and elucidating the heat adaptation mechanism in T. castaneum larvae.