Data from various studies have indicated that elevated temperatures damage fertility by affecting biochemical reactions in the testes. However, the specific mechanism of heat exposure leading to the damage and death of spermatogenic cells such as GC-2 spermatocyte cell line has not been well elucidated. The results in this study showed that the testis of mice had different degrees of damage with the increase of temperature. Glucocorticoid (GC) analysis showed that high-temperature treatment leads to an abnormal ratio of female-to-male offspring in mice after heat exposure. In in vitro cellular experiments, it was experimentally demonstrated that GC-2 spermatocyte cells were most severely damaged 6 h after heat exposure. Transcriptome sequencing yielded activating transcription factor 3 (Atf3). Experiments can detect the increase of Atf3 expression after elevated temperatures led to the decrease of the downstream solute carrier family 7-member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression in vitro and in vivo validation, at same time, reactive oxygen species (ROS) accumulation was also detected in the treated cells, which triggered ferroptosis. This study explained the underlying causes of low male fertility caused by heat exposure.