Introduction: Diazepam is a commonly used member of benzodiazepines. Little attention seems to have been paid to the effects of diazepam on endocrine glands. Omega-3 fatty acids exhibit significant antioxidant and anti-inflammatory effects on biological systems. Objectives: To throw light on the effect of diazepam on the adrenal cortex and the ameliorative effect of Omg-3 on the adrenal cortex. Materials and Methods: Fifty adult male albino rats were allocated randomly into three classes: Group I (control group) subdivided into three equal subgroups. Group II (diazepam treated group), Group III (diazepam + omg-3 treated group). After 28 days, blood samples were obtained from all groups to measure serum ACTH and cortisone. Tissue samples from adrenal cortex were obtained for evaluating adrenal malondialdehyde, glutathione, and superoxide dismutase. The adrenal cortex were undergone tissue processing for light and electron microscopic analysis. Morphometerical and statistical analysis were done. Results: Diazepam treated group exhibited significantly elevated levels of serum ACTH and cortisone, elevated levels of MDA, and decreased values of SOD and GSH compared to the control group. Histopathological examination showed thickened capsule, congested blood vessels, many cells with vacuolated cytoplasm, increased lipid droplets, Dilated sinusoids and intrasinusoidal macrophages, many lysosomes, electron-dense mitochondria, dilated SER and dark nuclei. Increased collagen fibers in the capsule and weak PCNA immunostaining. However, concomitant administration of omg-3 had an ameliorating effect on these changes. Conclusion: Omega 3 exerted ameliorating effects against diazepam-induced damage of the adrenal cortex. Therefore, it will be recommended to add supplement of omega-3 to the patients treated with diazepam.