The association between the clinical use of nitroglycerin (NTG) and migraine suggests NTG as an animal model trigger for migraine. NTG-induced hyperalgesia in rats has been extensively used as a migraine model for pre-clinical research. Pregabalin is an anti-epileptic drug and may play a role in the preventive treatment of migraine; however, the mechanism of this action remains to be clarified. Herein, we performed the present study to investigate the effect of pregabalin on the NTG-induced hyperalgesia in rats. Sixty male Sprague-Dawley rats were divided equally into six groups. Thirty minutes before NTG injection, the rats were pretreated with pregabalin. von Frey hair testing was employed to evaluate tactile sensitivity. Enzyme-linked immunosorbent assay was used to analyze plasma calcitonin gene-related peptide (CGRP) levels in the jugular vein. Immunohistochemistry was applied to detect c-Fos-immunoreactive neurons and western blot was performed to detect c-Fos protein expression in trigeminal nucleus caudalis (TNC). We found that pregabalin pretreatment alleviated the NTG-induced hyperalgesia. Moreover, pregabalin suppressed peripheral CGRP release, c-Fos-immunoreactive neurons and the protein expression of c-Fos in TNC as well. These data suggest that pregabalin could alleviate the NTG-induced hyperalgesia. Further studies are required to determine the mechanisms of action for this effect.