Total flavonoids of Epimedium (TFE) is the main active composition of Epimedium that has been used to treat male reproductive problems. The present aim was to investigate the protective effects of TFE on male mice reproductive system against cyclophosphamide (CP)-induced oxidative injury. The animals were treated with CP to make testicular injury model and the protective effects of TFE were observed. In the CP-treated group, testicular and epididymal weights, sperm count and motility significantly decreased relative to the control group (P < 0.05 and P < 0.01, respectively). Compared with the CP-treated group, TFE (200 and 400 mg/kg) treated mice increased testicular weights by 21.6% and 28.4% (P < 0.05), sperm counts by 81.7% and 148.3% (P < 0.01) and sperm motility by 47.2% and 61.3% (P < 0.01). Meanwhile, the CP-treated group showed enhancement of lipid peroxidation leading to testicular reproductive toxicity. TFE restored these oxidative damages by up-regulating the expression of antioxidant enzymes, especially SOD3 and GPX1. TUNEL assay and histopathological observations provided supportive evidence for above results, and when the dose of TFE increased, the aforesaid improvement became more and more strong. These results demonstrated that TFE exerted beneficially protective effects on the structural and functional damage of male mice reproductive system and reduced apoptosis in spermatogenic cells by inhibiting CP-induced oxidative stress. Copyright © 2013 John Wiley & Sons, Ltd.