Environmental heat stress (HS) impairs reproductive efficiency in farm animals. This study investigated, for the first time, how the melatonin and melatonin nanoparticles treatment affected the testicular hemodynamics, testicular volume, echotexture [Pixel intensity (PIX) and integrated density (IND)], scrotal circumference, serum concentration of testosterone (T), estradiol (E2), nitric oxide (NO), and total antioxidant capacity (TAC) in prepubertal Ossimi ram lambs in hot climatic conditions. The lambs undergoing examination had a temperature humidity index (THI) of 87.05 ± 1.70, indicating severe HS condition. Fifteen prepubertal Ossimi ram lambs were exposed to a single s.c injection of either nano melatonin (nano melatonin group; 20mg/ram; n = 5) or melatonin suspended in two ml of corn oil (melatonin group; 40mg/ram; n = 5) or two ml of corn oil (control group; n = 5). Blood collection and ultrasonographic assessment of the testes and supratesticular arteries (STAs) were conducted immediately before treatment (W0) and once weekly for six successive weeks after nano melatonin and melatonin injection (W1-W6). Results revealed decreases (P < 0.05) in the Doppler indices (resistive index; RI and pulsatility index; PI) of the testicular arteries at most time points of the study in the nano melatonin and melatonin groups. PIX of testicular parenchyma was significantly increased (P ˂ 0.05) in the treated groups compared to the control one. IND of testicular parenchyma increased significantly in the nano melatonin group compared to the melatonin and control groups. Testicular volume and scrotal circumference significantly increased (P < 0.05) in nano melatonin and melatonin groups compared to the control one. T concentration did not significantly (P > 0.05) change in the treated groups compared to the control group. E2, NO, and TAC concentrations increased (P < 0.05) in the treated groups compared to the control one. In conclusion, this study extrapolated that administrations of melatonin or nano melatonin can ameliorate the effects of environmental HS in prepubertal Ossimi ram lambs with a more protective effect and lower dose of nano melatonin.