Background: Cyclophosphamide is used for the treatment of malignant and non-malignant diseases, but, it induces oxidative damage and disturbance in the antioxidant defense system. Zinc oxide nanoparticles (ZnO NPs) are used in biomedical applications and consumer products. ZnO-NPs are protected cell membranes against oxidative damage, decrease free radicals and malondialdehyde (MDA) levels, and increase the antioxidant enzyme levels. Objectives: The present aimed to evaluate the ameliorative effect of Zn-O nano-particles on oxidative damage and disturbance in the antioxidant defense system induced by cyclophosphamide in male albino rats. Materials and Methods: 24 adult male albino rats were randomly divided into 4 groups (6 rats of each). Group I (Control group): Received 0.2 ml saline /day i.p. injection for 14 days (day by day), group II, (nZnO group): Received nZnO (5 mg/kg/day) b.w., intraperitoneally for 14 days, Group III (CP group): Received CP (20 mg/kg/day) b.w, day by day for 14 days by intraperitoneal injection, Group IV (CP + ZnO NPs group): Received nZnO group: Received nZnO (5 mg/kg/day) b.w., intraperitoneally for 14 days, plus CP (20 mg/kg/day) b.w., day by day for 14 days by intraperitoneal injection. After 24-hr from the last treatment, all animals were anesthetized using light ether. Blood, lungs, and liver samples were taken and prepared for biochemical measurements. Results: Individual treatment of zinc oxide nanoparticles and CP induced liver cytochrome b5, cytochrome C reductase, and glutathione S-transferase (GST) compared to the control group, while CP increased P450. The combination of nZnO and CP prevents the elevation of cytochrome b5, P450, cytochrome C reductase, and GST compared with the CP treated group. Zinc oxide nanoparticles and CP increased liver thiobarbituric acid reactive substances (TBARS). The combination of nZnO and CP prevents the changes in TBARS concentrations compared with the CP. Injection of CP to rats reduced the activities of serum glutathione reductase (GR) and catalase (CAT) as compared with the control group. However, combination treatment of rats with nZnO and CP increased the activities of these enzymes compared with those treated with CP alone. Zinc oxide nanoparticles and CP increased serum and lung TBARS, while decreased glutathione (GSH) concentration compared to the control group, with more pronounced changes by CP. The combination of nZnO and CP prevents the changes in TBARS and GSH concentrations compared with the CP. Conclusion: It can be concluded that CP induced oxidative stress and disturbance in the antioxidant defense system. Treatment of rats with zinc oxide nano-particles and CP together attenuated the oxidative damage and disturbance in the antioxidant defense system induced by CP. So, Patients treated with CP advised to take nZnO to prevent the side effects of chemotherapy. Further studies are necessary to evaluate the amelioration effect nZnO and other nano-particles against oxidative stress induced by CP in different doses and experimental models.