Testicular ischemia-reperfusion is accompanied by elevated production of reactive oxygen species. It has been reported that reactive oxygen species are highly reactive to cellular carbohydrates, DNA, lipids and proteins, and result in testicular ischemia-reperfusion injury. Gastrodin is the principal active ingredient isolated from the medicinal plant Gastrodia elata Blume and has anti-oxidative stress effect. In this study, we explored the potential protective activity of gastrodin in rat testicular ischemia-reperfusion injury model and underlying mechanism. Male rats were randomized into 3 groups (sham control, testicular ischemia-reperfusion injury, testicular ischemia-reperfusion injury along with gastrodin injection) (n = 20). Testicular ischemia-reperfusion injury group received 2-hour period of left testicular torsion (720°, counterclockwise) and 4-hour or 3-month period of testicular detorsion. At the onset of testicular detorsion, gastrodin-treated rats were given 100.00 mg kg-1 gastrodin by intraperitoneal route. Following testicular detorsion, testicular tissues were collected for enzymatic activity analysis, oxidative stress evaluation and histopathological examination. The ipsilateral testicular xanthine oxidase activity (source of reactive oxygen species production) and malondialdehyde level (a precise biomarker of reactive oxygen species) were significantly increased in testicular ischemia-reperfusion injury group versus sham control group, while testicular spermatogenic function was decreased. Furthermore, gastrodin administration reduced xanthine oxidase activity and malondialdehyde level in ipsilateral testicular tissue, while improving testicular spermatogenic function. Consequently, it is suggested that gastrodin plays a protective role in testicular torsion/detorsion-induced ischemia/reperfusion injury through inhibiting xanthine oxidase activity to decrease reactive oxygen species formation.