The current study aimed to examine the effects of nanographene oxide on the testes. A total of 48 male albino rats were randomly divided into 6 groups. The first, second, third, fourth, and sixth groups were treated with graphene oxide nanopowder at 20, 30, 40, 50, and 60 mg/kg concentrations, respectively. The sixth group was considered the control group. The results indicated a significant decrease in the average testis weight of rats treated with different nanographene oxide dosages, compared to the control group. There was also a significant decrease in the level of FSH and testosterone of treated rats with nanographene oxide, while there was no significant difference in the level of LH hormone when compared to the control group. The histological examination of the testes in the treated rats indicated hemorrhage, decreased sperm count, decreased thickness of the tubular epithelium, dissociation of connective tissue between the seminiferous tubules, in addition to hematological congestion, necrosis of the tubular epithelium, divergence of the seminal tubules, absence of sperm, shattering of the seminal tubule wall and degeneration sperm-forming cells and edema formation. Using the transmission electron microscope, the findings revealed a range of cellular changes, such as the presence of two-headed spermatids, the destruction of the nucleus membrane, spermatoblasts, the destruction of the cell membrane, and the denting of the nucleus membrane. It can be concluded that the nanographene oxide at 20-60 mg/kg concentrations can have harmful effects on spermatogenesis and normal function testis in rats.