Objectives: Spermatogenesis, in which cell regeneration continues, can be affected by environmental, chemical, psychological factors or various diseases. There is conflicting information in the literature about the effect of isotretinoin, which is widely used in acne treatment, on testes and spermatogenesis. Therefore, we planned a rat study to evaluate the long-term efficacy of oral isotretinoin on testicular tissues and spermatogenesis. Materials and methods: The Group 1 (n = 6) 7.5 mg/kg/day and the Group 2 (n = 6) received isotretinoin at a dose of 30 mg/kg/day dissolved in sunflower oil, the Sham Group (n = 6) received only sunflower oil by gavage, and the control group (n = 6) received standard feed and water for four weeks. After the 4th week, all animals were fed with standard feed and water and followed for the next four weeks. At the end of the 8th week, all animals were sacrificed under deep anesthesia. Seminiferous tubule diameters, epithelial thickness, apoptotic index, sperm number and motility recorded. Results: Sperm count, motility, vitality, diameter of seminiferous tubule and germinal epithelium thickness were decreased and apoptotic index increased in the groups received isotretinoin. There was no significant difference between the groups in terms of testosterone levels. Conclusions: We consider that further comprehensive studies, including human clinical trials, should be conducted to examine the negative effects of isotretinoin on spermatogenesis in the long term especially when there is a need using isotretinoin in men for various reasons and to eliminate the contradictions in the literature in this regard.