The present study evaluated the protective effects of royal jelly (RJ) on methotrexate (MTX)-induced testicular damage in rats, focusing on oxidative stress and autophagy. Methotrexate, a folic acid analogue used in cancer and autoimmune treatments, impairs spermatogenesis via oxidative stress and apoptosis. Twenty-four male Wistar rats were randomized into four groups: Control (normal saline, 35 days), MTX (0.30 mg kg-1, gavage, three times per week, 35 days), MTX + RJ (0.30 mg kg^-1 MTX + 0.10 mg kg^-1 RJ, gavage, three times per week, 35 days), and RJ (0.10 mg kg^-1, gavage, three times per 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 diameter, epithelial thickness, tubular differentiation index (TDI) and spermiogenesis index. Co-administration of RJ significantly improved seminiferous tubule morphology, diameter, epithelial thickness, TDI and spermiogenesis index. 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. 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. These findings suggested that RJ mitigated MTX-induced testicular damage by reducing oxidative stress and autophagy, thereby, preserving spermatogenesis and testicular integrity.