Diabetes mellitus is a metabolic disease affecting various organs, including the spleen and is characterized by chronic hyperglycemia. Oxidative and inflammatory stress are key mediators in the development of spleen damage caused by diabetes. This study aimed to examine the splenoprotective effect of hesperidin and the mechanisms underlying its capacity to reduce oxidative stress and inflammation-mediated spleen damage in diabetes. The diabetic rats used in this study were induced with a 65 mg per kg body weight of streptozotocin. This was followed by 4 weeks of continuous daily dosage of hesperidin treatment at 100 mg/kg body weight. The results showed that hesperidin improved spleen weight and histopathological alterations in the diabetic rats. The hesperidin-treated diabetic group showed a marked induction of SOD and GPx enzymes and moderated malondialdehyde level. This was in addition to an obvious decrease in the levels of TNF-α and NF-ᴋB in the diabetic rat spleen. Through a remarkable upregulation in Bcl-xL and downregulation in Bax and cleaved caspase-3 proteins, hesperidin supplementation rescued splenic cell apoptosis in the diabetic rats. These findings demonstrate the effectiveness of hesperidin in helping regulate Bcl-2 family proteins and inhibiting the oxidative stress and inflammatory status of hyperglycemia-mediated spleen apoptosis. PRACTICAL APPLICATIONS: Diabetes-related spleen damage increases immune dysfunction, which often results in the heightened risks of infection, morbidity and mortality in diabetic patients. In this work, hesperidin was used in the treatment of rats with diabetes-induced splenic damage. The results were highly encouraging with hesperidin consistently presenting beneficial antioxidant and anti-inflammatory qualities and splenoprotective effect. Research outcomes support the notion that hesperidin treatment could be considered a good strategy for the prevention of diabetic complications in the spleen.