Introduction: Spreading depression (SD) is a pathophysiologic phenomenon followed by a transient reduction in neuronal activity. Several studies indicate that SD plays a role in some neurological disorders. The present study is aimed to investigate the effect of SD on the expression of GABA Aα receptors in the mesencephalic trigeminal nucleus. Materials and Methods: Animals were randomly classified into three control, sham, and experimental groups. Animals were anesthetized and during the stereotaxic surgery, recording electrodes and the injection cannula were implanted over the cortex. In the experimental group, KCl solution was injected four times once a week into the neocortex to induce repetitive SD. In sham group Ringer solution was injected and control group received no intervention. At the end of this stage, the animals were perfused and the brains were removed. Consequently, histological examination of the brains and immunohistochemical staining method were used to investigate the mean number of dark neurons as well as the expression of GABA Aα receptors and GAD65 enzyme. Results: The mean number of dark neurons significantly increased in the experimental group compared to the sham and control groups. Expression of GABA Aα receptors and GAD65 enzyme in mesencephalic trigeminal region decreased significantly after SD in the experimental group compared to the other groups. Conclusion: Our findings indicate that SD modulates inhibitory receptors in mesencephalic trigeminal region. This may be important in treatment of cephalalgia.