Simple SummaryMitotane (MTT) is the treatment of choice for adrenocortical carcinoma. Male hypogonadism is often diagnosed in male patients treated with this drug. This research aims to consider possible side effects induced by MTT in the testis and a hypothetical detrimental effect deriving from androgenic deficiency. Furthermore, considering the increased potentiality of treatment for adrenocortical carcinoma, we want to provide male patients of childbearing age, as in other oncological diseases, the opportunity to consider a sperm cryopreservation program.AbstractBackground: Clinical evidence has shown frequent hypogonadism following mitotane (MTT) treatment in male patients with adrenocortical carcinoma. This study aimed to evaluate the impact of MTT on male gonadal function. Methods: Morphological analysis of testes and testosterone assays were performed on adult CD1 MTT-treated and untreated mice. The expression of key genes involved in interstitial and tubular compartments was studied by real-time PCR. Moreover, quantitative and qualitative analysis of spermatozoa was performed. Results: Several degrees of damage to the testes and a significant testosterone reduction in MTT-treated mice were observed. A significant decline in3βHsd1andInsl3mRNA expression in the interstitial compartment confirmed an impairment of androgen production.Fsh-RmRNA expression was unaffected by MTT, proving that Sertoli cells are not the drug’s primary target. Sperm concentrations were significantly lower in MTT-treated animals. Moreover, the drug caused a significant increase in the percentage of spermatozoa with abnormal chromatin structures. Conclusion: MTT negatively affects the male reproductive system, including changes in the morphology of testicular tissue and reductions in sperm concentration and quality.