Male reproductive dysfunctions and infertility are the common consequences of overt diabetes. Available evidence support oxidative stress to be the underlying mechanism for the manifestation of testicular complications during diabetes. In the present study, we assessed the attenuating effects of Withania somnifera root extract (WS) on diabetes-induced testicular oxidative disturbances in prepubertal rats. Four-week-old prepubertal rats were assigned into nondiabetic control, streptozotocin (STZ)-treated and STZ+WS supplemented (500 mg/kg b.w./d, oral, 15 days) groups. Experimental diabetes was induced by a single intraperitoneal injection of STZ (90 mg/kg b.w). Terminally, all animals were killed, and markers of oxidative stress were determined in the testis cytosol and mitochondrial fraction. Severe hyperglycemia and regression in testis size were apparent in diabetic rats. A decline in antioxidant defenses with subsequent elevation in the generation of reactive oxygen species and lipid peroxidation was discernible in testis cytosol and mitochondria of diabetic prepubertal rats, which was significantly reversed by WS. However, there was partial restoration of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and 3-beta hydroxysteroid dehydrogenase activities in testis of diabetic prepubertal rats administered with WS. Taken together, data accrued suggest the potential of WS to improve diabetes-induced testicular dysfunctions in prepubertal rats.