Increased DNA damage in testis is considered as a major factor for ageing-related dysfunction. Total flavonoids of Epimedium (TFE), the main active compositions of Epimedium, have been used to treat sexual dysfunction and delay ageing. However, whether TFE could improve ageing-related testicular dysfunction remains unknown. Therefore, we investigated the protection effects of TFE and its mechanisms of action in a naturally ageing rat model. Eighteen-month-old SD rats were randomised to receive either vehicle or TFE (10 and 20 mg/kg). Nine-month-old SD rats were used as adult controls. Morphology, protein expression and immunohistochemistry were determined. Compared with adult control group, intragastric administration of TFE for 6 months significantly improved testicular morphology, increased the activities of SOD and decreased the levels of MDA of testis. In addition, TFE decreased γH2AX expression levels and γH2AX focal formation in spermatogonia and primary spermatocyte with concomitant downregulation of 8-OHdG levels. Furthermore, TFE inhibited p-P53/p21 and chk1/chk2 expression levels. Collectively, TFE effectively reduce oxidative DNA damage in testis of ageing rats via a p53-dependent pathway. Thus, inhibition of oxidative DNA damage is likely to represent a promising strategy for restoration of ageing-related testicular dysfunction.