As a common metabolic disorder, hyperglycemia (HG) affects and disrupts the physiology of various systems in the body. Transplantation of mesenchymal stem cells (MSCs) has been used to control the complications of disease. Most of the therapeutic properties of MSCs are attributed to their secretome. This study aimed to investigate the effects of conditioned media extracted from sole or caffeine pre-treated bone-marrow-derived MSCs on hyperglycemia-induced detrimental impact on some aspects of reproduction. The HG was induced by intraperitoneally injection of streptozotocin (65 mg/kg) and nicotinamide (110 mg/kg). Twenty-four male Wistar rats (190 ± 20 g) were divided into control, HG, and the hyperglycemic groups receiving conditioned media of proliferated MSCs solely (CM) or MSCs pre-treated with caffeine (CCM). During the 49-day treatment, body weight and blood glucose were measured weekly. Finally, HbA1c, spermatogenesis development, sperm count, morphology, viability, motility, chromatin condensation, and DNA integrity were examined. Also, testicular total antioxidant capacity (TAC), malondialdehyde, sperm fertilization potential, and pre-implantation embryo development were evaluated. A one-way ANOVA and Tukey's post-hoc tests were used to analyze the quantitative data. The p < 0.05 was considered statistically significant. The CM and with a higher efficiency, the CCM remarkably (p < 0.05) improved body weight and HG-suppressed spermatogenesis, enhanced sperm parameters, chromatin condensation, DNA integrity, and TAC, reduced HbA1c, sperm abnormalities, and malondialdehyde, and significantly improved pre-implantation embryo development versus HG group. The conditioned media of MSCs solely (CM) and more effectively after pre-treatment of MSCs with caffeine (CCM) could improve spermatogenesis development, sperm quality, pre-implantation embryo development, and testicular global antioxidant potential during hyperglycemia.Keywords: Fertilization; Hyperglycemia; Mesenchymal stem cells; Sperm; Spermatogenesis.