Abstract Background Chinese medicine Wuzi Yanzong pill (WZYZP) was firstly documented in ancient Chinese medical works “She Sheng Zhong Miao Fang” by Shi-Che Zhang in 1550 AD. The traditional herbal formula is widely used in treating nephrasthenia lumbago, prospermia, erectile dysfunction and male sterility. The present study was to explore the effects of WZYZP on ionizing irradiation-induced testicular damage in mice. Methods The pelvic region of male mice was exposed to X-rays for inducing testicular damage. The effects of WZYZP on testicular damage were evaluated in terms of testes weight, sperm quantity and motility, testes oxidative status and serum hormone levels. The alterations in testicular structure were examined by hematoxylin-eosin staining. Additionally, changes in proliferating cell nuclear antigen (PCNA) expression of testes were explored by western blot. Results Pelvic exposure to x-ray induced reduction in testes weight and sperm quality, along with oxidative stress and abnormal testicular architecture in testes. Oral administration of WZYZP for 3 weeks markedly increased testes weight, sperm quantity and motility, and attenuated testicular architecture damage. Meanwhile, WZYZP treatment significantly reversed the reduction of serum testosterone, and decreased testes malondialdehyde (MDA) and Oxidative stress index (OSI) relative to the radiated mice. Additionally, WZYZP effectively prevented the downregulation of PCNA expression in testes induced by x-ray irradiation. Conclusion These findings suggest WZYZP exhibits ameliorating effects against ionizing irradiation-induced testicular damage in mice, which may be related to its antioxidation.