Background Iron oxide nanoparticles, especially nano-magnetite, are promising candidates for use in a variety of applications. The present study aimed to investigate the effect of nano-magnetite on the reproductive health of female Wistar rats. Twenty-one adult female rats were divided into three groups: Group 1 served as the control group, Group 2 received a low dose of 5 mg/kg of nano-magnetite, and Group 3 received a high dose of 10 mg/kg of nano-magnetite. For 30 days, rats were intraperitoneally injected three times per week. The main findings Revealed that nano-magnetite did not induce a change in body weight or absolute as well as relative reproductive organs weight. Nano-magnetite nanoparticles influenced the reproductive serum hormone levels as well as imbalanced the ovarian and uterine malondialdehyde and total antioxidant activity. After nano-magnetite nanoparticle injection, the histopathological examination revealed apoptosis of granulosa cells of various types of follicles, degenerated corpora lutea, congested blood vessels, and uterine epithelial cells of uterine tissue showed a high level of apoptosis and inflammation. Immunohistochemistry studies demonstrated a significant increase in activated caspase-3 following nano-magnetite injection, indicating an increase in cell apoptosis. Conclusion This study demonstrated the negative effect of magnetite nanoparticle on reproductive health and increased the likelihood of infertility.