Introduction: Phenobarbital (PB) is a commonly used classical anticonvulsant drug. It is effective in treating partial tonic-clonic fits. Adverse effects of antiepileptic drugs on the endocrine glands were reported producing disturbed endocrine function. Aim of the work: Was to study the possible effect of phenobarbital administration on rats' different endocrine glands using histological, hormonal assay and morphometric studies. Material and Methods: Fifty adult male albino rats were used, divided into 3 groups; A control group (I); consisted of 10 rats, phenobarbital group (II); contained 20 rats, each rat received 7.5 mg phenobarbital sodium/100gm body weight orally for 30 days. PB withdrawal group (III); consisted of 20 rats, each received the same PB dose for 30 days orally and have been left without treatment for another 15 days. Results: Examination of the pituitary, thyroid and adrenal gland sections of PB group showed dilatation and congestion of blood sinusoids, cytoplasmic vacuoles and irregularity of nuclei of pituitary secretory cells, marked decrease in both follicular size and colloid contents with destruction of the microvillus border of the thyroid follicles, increase in thicknesses of both adrenal cortex and medulla with disruption of the architecture of adrenal cortical layers. Examination of the PB withdrawal group showed evidence of improvement in the three glands as compared to the PB group. Conclusion: Phenobarbital can cause harmful histopathological changes on the thyroid gland, adrenal cortex and pituitary gland and its withdrawal resulted in an improvement in these deleterious effects to a lesser extent.