Background Tramadol is a centrally acting opioid analgesic drug that is widely used in the treatment of pain. It is currently the most prescribed opioid in the world, so its abuse, dependence, toxicity, and tramadol-related deaths have increased. Aim This study aimed to evaluate the histological effect of tramadol on the seminiferous tubules of testis of adult albino rat and assess the effect of its withdrawal. Materials and methods A total of 52 adult male albino rats were used and divided into two main groups: the first served as control, and the second was treated with tramadol and subdivided into four subgroups. The first and third subgroups received tramadol once daily by gastric tube in a dosage of 40 mg/kg for 4 and 6 weeks, respectively, whereas the second and the fourth (recovery subgroups) received tramadol in a dosage of 40 mg/kg once daily for 4 and 6 weeks, respectively, and then kept without treatment for another 4 weeks. Specimens from the testes were processed for light and electron microscopic examination. Immunohistochemical study was carried out. Morphometric study was also done, and statistical results were analysis. Results Specimens from tramadol-treated animals showed obvious structural changes. Some spermatogenic cells appeared disorganized with wide intercellular spaces. Many spermatogonia showed vacuolated cytoplasm and deeply stained nuclei. Ultrastructurally, Sertoli cells showed cytoplasmic vacuoles. Some primary spermatocytes appeared with concentric lamellar formations. The immunohistochemical study showed a highly significant decrease in Bcl-2 immunoreaction. In contrast, minimal changes were observed in the recovery groups, with a nonsignificant change in the immunoreaction. Conclusion Tramadol-induced histological and immunohistochemical effects on the seminiferous tubules of rat testis, so its abuse should be avoided except with medical prescription.