Migraine is one of the most common neurological disorders characterized by recurrent attacks of typically throbbing and unilateral headaches, affecting up to 20 % of the population worldwide. Despite the high prevalence and severity of this primary headache disorder, it remains to be a challenge to fully understand and treat migraine headaches. By characterizing and validating a mouse migraine model, this study aimed to investigate the functional contribution of PKC isoforms in migraine. In this study, we identified the presence of migraine-like ongoing pain in mice after chronic intermittent treatment with nitroglycerin (NTG). The peptide antagonist of calcitonin gene related peptide (CGRP) α-CGRP (8-37), but not topiramate nor sumatriptan, effectively blocked ongoing pain and elicited pain relief-induced CPP in NTG-treated mice. Prominent activation of PKCδ was observed in chronic NTG-treated mice. Functional inhibition of PKCδ significantly attenuated ongoing spontaneous pain in chronic NTG-treated mice. Furthermore, we recapitulated the NTG-triggered migraine behavior in wild-type, but not in PKCδ-null mice. In response to repeated administration of NTG, ongoing spontaneous pain was not developed in mice lacking the specific PKC isoform. This study identified the presence of ongoing pain in mice treated with nitroglycerin, a known human migraine trigger that closely resembles the common manifestation of spontaneous migraine attacks in humans. These findings demonstrated a critical regulatory role of PKCδ in migraine pathophysiology, which may offer new pharmacological targets for anti-migraine treatment.