The present study evaluates the protective effects of royal jelly (RJ) on methotrexate (MTX)-induced testicular damage in rats, focusing on oxidative stress and autophagy. MTX, a folic acid analogue used in cancer and autoimmune treatments, impairs spermatogenesis via oxidative stress and apoptosis. Twenty-four male Wistar rats were divided into four groups: control (normal saline, 35 days), MTX (0.3 mg/kg, gavage, 3 times/week, 35 days), MTX+RJ (0.3 mg/kg MTX + 0.1 mg/kg RJ, gavage, 3 times/week, 35 days), and RJ (0.1 mg/kg, gavage, 3 times/week, 35 days). After 35 days, rats were euthanized, and testicular tissue was analyzed via histopathology, immunohistochemistry for LC3-I/II expression in germ cells, and qRT-PCR for mRNA expression of autophagy-related genes (Beclin-1, Atg7, LC3-I). Histopathological findings revealed that MTX caused severe interstitial edema, coagulative necrosis, and disrupted spermatogenesis, with reduced seminiferous tubule (ST) diameter, epithelial thickness, tubular differentiation index (TDI), and spermiogenesis index (SPI). Co-administration of RJ significantly improved ST morphology, diameter, epithelial thickness, TDI, and SPI (p<0.05). Immunohistochemistry showed a significant increase in LC3-I/II+ germ cells (spermatogonia, spermatocytes, spermatids) in the MTX group, which was markedly reduced in the MTX+RJ group (p<0.05). Similarly, qRT-PCR analysis demonstrated elevated mRNA levels of Beclin-1, Atg7, and LC3-I in the MTX group, which were significantly reduced in the MTX+RJ group (p<0.05). These findings suggest that RJ mitigates MTX-induced testicular damage by reducing oxidative stress and autophagy, thereby preserving spermatogenesis and testicular integrity.