Heat stress is an important influence on the male reproductive organs. Therefore, the effects of heat stress on genes or pathways related to the reproductive system of male mice were experimentally explored in this paper to further determine the effects of heat stimulation on mammals. Herein, models of heat-exposed mouse testicular tissue and heatexcited cells were successfully established. Many scorched vesicles were found after heat excitation of testis supporting cells, testicular mesenchymal (TM4) cells. Western blot, in situ terminal deoxynucleotide transferase dUTP Nick end labeling (TUNEL) and transmission electron microscopy showed that membrane rupture, mitochondrial damage and autophagic vesicles occurred in TM4 cells after thermal excitation. The N-segment fragment of the associated protein shear was increased, and the TUNEL result was positive. In conclusion, thermal excitation induced apoptosis and scorch death in TM4 cells. Thus, the Hippo pathway and apoptosis-related pathway were significantly enriched after heat stimulation in mouse testis, and the scorch death effect in TM4 cells was induced by heat excitation.