Cisplatin exposure represents a significant fertility problem for childhood cancer. In this study we examined the possible therapeutic role of Zinc oxide nanoparticles (ZnO-NPs) on Cisplatin (Cis) induced impairment in the spermatogenesis initiation during puberty. Seventy-two male rats aged 30 days were distributed into four equal groups: Control group; ZnO-NPs group (intraperitoneal i.p. injected with 5 mg/kg ZnO-NPs once a week for eight weeks); Cis group (i.p. injected with a single dose of 5 mg/kg); ZnO-NPs + Cis group (ZnO-NPs injection 2 hrs before Cis). Each group was subdivided into three groups and was sacrificed 7, 30 and, 60 days after cisplatin induction, which considered prepubertal at 37-day-old, productive at 60-day-old, and completely adult at 90-day-old. Biochemical, molecular, immunohistochemical, and ultrastructural examinations were studied on the testicular tissues and sperm samples. Group treated with Cis showed a decrease in the antioxidant activity and an increase in the reactive oxygen species (ROS), which in turn caused disruption in blood-testis barrier (BTB) proteins in the three different rat ages, and sperm DNA damage in the adult rats compared to control group (p < 0.05). Moreover, alterations in the structural and the ultrastructural morphology of the testis were observed compared with the control at 37, 60 and 90 days old rats. ZnO-NPs administration to Cis group manifested a significant decrease in the ROS that restored the BTB proteins, enhanced the architecture of the testis in the three different rat ages, and increased sperm DNA integrity in the adult rats. Zinc oxide nanoparticles could restore the male reproductive capacity in the adult rats after induction of Cis in the prepubertal period by promoting spermatogenesis.