In the present study, we investigated the protective effect of magnolin (MAG) against oxidative stress induced by cyclophosphamide (CP) and its role in the signaling pathway. Rats were administered MAG (1 mg/kg, i.p.) for 14 days and CP (75 mg/kg, i.p.) on the 14th day. CP administration increased tissue damage, as evidenced by elevated levels of transaminases (aspartate and alanine), alkaline phosphatase, and renal parameters (blood urea nitrogen and creatinine). Additionally, 8-hydroxy-2'-deoxyguanosine and malondialdehyde levels were increased, whereas glutathione levels, along with catalase and superoxide dismutase activities, decreased in CP-treated rats. CP also down-regulated the expression of and , while up-regulating α and in liver and kidney tissues. In addition, CP treatment caused histopathological changes in heart, lung, liver, kidney, brain, and testis tissues. Treatment with MAG improved biochemical and oxidative stress parameters and prevented histopathological changes in CP-treated rats. Moreover, MAG suppressed the expression of inflammatory cytokines and apoptosis markers. In conclusion, MAG effectively prevented CP-induced toxicity by reducing oxidative stress, inflammation, and apoptosis, with its protective efficacy associated with the up-regulation of signaling.