Objective To investigate the role of protein kinase Cγ (PKCγ), N-methyl-D-aspartic acid receptor 1 (NMDAR1), and phosphorylated NMDAR1 in the pathogenesis of migraine in a rat model of nitroglycerin-induced migraine.Methods A total of 30 adult female Sprague-Dawley rats were randomly divided into control group, model group, and intervention group, and each of the latter two groups was further divided into ictal period group and interictal period group, with 6 rats in each group. The rats in the model group and the intervention group were used to establish a model of migraine according to the Tassorelli Cristina method, and those in the control group were given normal saline. The rats in the intervention group were given flunarizine 2 ml (0.5 mg/kg) daily by gavage, and those in the control group were given normal saline 2 ml daily by gavage. The rats were decapitated at 2 hours (the ictal period group) or on day 4 (the control group and the interictal period group) after the fifth time of model establishment, and brainstem tissue samples were collected. RT-PCR was used to measure the mRNA expression of PKCγ and NMDAR1, and Western blot was used to measure the protein expression of PKCγ, NMDAR1, and phosphorylated NMDAR1.Results There were no significant differences in the mRNA and protein expression of PKCγ and NMDAR1 in brainstem tissue between the control group and the other four groups (P>0.05). Compared with the control group, the model group had a significant increase in the protein expression of PKCγ-dependent phosphorylated NMDAR1 (P<0.05) and the intervention group had a slight increase (P>0.05). There was no significant difference in the protein expression of phosphorylated NMDAR1 between the model group and the intervention group, as well as between the ictal period group and the interictal period group (P>0.05).Conclusions The development and progression of migraine might be associated with the upregulated protein expression of PKCγ-dependent phosphorylated NMDAR1 and may not be associated with the mRNA and protein expression of PKCγ and non-phosphorylated NMDAR1. Flunarizine can prevent the attack of migraine by interrupting the vicious circle of the PKC-NMDAR positive feedback loop.