Background: Testicular torsion (the most common cause of testicular ischemia) is one of the urologic emergencies occurring frequently in neonatal and adolescent period. Although reperfusion of the testis is the key treatment after ischemia, it enhanced the formation of reactive oxygen species (ROS) that result in testicular damage and cell apoptosis. The effect of obestatin on testicular ischemia-reperfusion injury has not been evaluated previously. Aim of the work: This study was delineated to investigate the potential protective effect of obestatin on testicular ischemia/reperfusion (I/R) injury in rats. Materials and methods: 30 healthy adult male albino rats weighting 194-217 g were involved and divided into 3 equal groups, group (I): sham operated control group, group (II): testicular ischemia reperfusion (I/R) group and group (III): obestatin treated ischemia reperfusion (I/R-obestatin) group; received obestatin (100 μg/kg) intravenously 15 min before the testicular detorsion (reperfusion). Serum testosterone level was measured and orchiectomy was performed 6 hours after testicular detorsion and examined histopathologically and immunohistochemically for evaluation of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins expression. Testicular malondialdehyde (MDA), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), caspase-8 and caspase-3 activities were also evaluated. Results: This study demonstrated that administration of obestatin prior to detorsion significantly increased the reduced serum testosterone level and attenuated the testicular tissue damage observed in I/R group. It also reversed the increased MDA, TNF-α and IL-1β levels and significantly enhanced the antioxidant enzymes activities in the ipsilateral I/R testis. Moreover, it improved the significant high levels of caspase-8 and caspase-3 and alleviated the increased Bax and the reduced Bcl-2 proteins expressions noticed in I/R testis. Conclusion: Obestatin has a protective effect against testicular I/R injury which can be attributed to its anti-oxidant, anti-inflammatory and anti-apoptotic properties Key words: obestatin, reperfusion injury, testis, oxidative stress.