Abstract: Objective To evaluate the stress response in mouse testes following a single mild transient scrotal heat exposure (43 degrees C for 15 min). Methods Twenty-four male mice were randomly divided into four groups with six mice per group. Mice in scrotal hyperthermia group were subjected to a single heat stress of 43℃(treated) for 15 min and killed at 0.5 h, 2 h, and 6 h after heat exposure. The control mice were immersed in a water bath maintained at 22℃ and killed at 6 h after heat exposure. Histopathological analyses of testes were performed by hematoxylin and eosin (H&E) staining. Testicular germ cell apoptosis was determined by terminal dUTP nick-end labelling (TUNEL). The expression of testicular caspase-3 protein was measured using western blot. Results As expected, scrotal hyperthermia markedly increased the inside diameter of seminiferous tubules and disturbed the array of spermatogenic cells. Meanwhile, Scrotal hyperthermia clearly induced cytoplasmic vacuolization, pyknosis of nuclei and multinucleate spermatogenic cells in seminiferous tubules in heat-treated mice, especially at 6 h after heating. Paralleled with testicular lesion, acute scrotal hyperthermia significantly increased testicular germ cell apoptosis comparing with control group. In addition, scrotal hyperthermia obviously upregulated the expression level of active caspase-3 in testes. Conclusions A single, acute scrotal hyperthermia significantly induce testicular histological impairment and germ cell apoptosis.