Introduction: It is now accepted that scrotal heat stress could adversely affect spermatogenesis. This high thermal condition can cause a reduced male fertility potential. Nowadays, insufficient research exists on the impact of transient scrotal hyperthermia on heat shock proteins 70 and 90 in murine subjects. In the current study, we investigated the effects of scrotal hyperthermia on the expression of heat shock proteins, stereological parameters, and semen quality in mice. Methods: In this examination, a total of 18 healthy adult male NMRI mice were divided equally into two groups: control and scrotal hyperthermia. Scrotal heat stress was induced by placing the lower parts of mice bodies into the water bath for three consecutive days (43°C, 20 min/day). Then, epididymis and testicular samples were collected for evaluation of sperm parameters, stereological study, mRNA, and protein expression of HSP70 and HSP90. Results: Our results revealed that scrotal hyperthermia could strikingly increase the level of mRNA and protein expression of HSP70 and HSP90 in the samples. In addition, stereological parameters and semen quality significantly decreased in transient scrotal hyperthermiainduced mice compared to the control group. Conclusion: Our research indicates that transient hyperthermia on the scrotum can lead to increased expression of HSP70 and HSP90 at both mRNA and protein levels, subsequently affecting male fertility.