Objective To investigate the effect of Zhengtian pills on P2X3 receptor expression in midbrain of rat model of migraine. Methods The Sprague-Dawle(SD) male rats were randomly divided into control group, migraine group and Zhengtian pills group, with 8 rats in each group. Rats of Zhengtian pills(ZTP) group were given 1.62 g/kg Zhengtian pills by gavage. Rats of control group and model group were given 1 mL 0.9% sodium chloride solution by gavage. The drug administration lasted for 7 days. In migraine group and ZTP group, migraine rat models were established by subcutaneous injection of nitroglycerin(NTC) (10 mg/kg) after 30 min of the last drug administration. Rats of control group were given 0.5 mL 0.9% sodium chloride solution by subcutaneous injection. Behavioural performances of the rats were observed, including the duration of ears pink and scratching head. Von-Frey hair was used to monitor the pain threshold of allodynia around whisker pad. Immunofluorescence and western blot were used to detect the expression of P2X3 receptors in midbrain of rats. Results Rats of migraine group and ZTP group were not significantly different in the appearing time of red ear and the beginning time of scratching head. ZTP group had earlier disappearing time of red ear and earlier ending time of scratching head than migraine group(P<0.001). After modeling, the pain threshold at each time point in the migraine group was significantly lower than that before modeling and the control group at the same time(P<0.05). The pain threshold of ZTP group decreased at 0.5 h after modeling(P<0.05), then it continued to rise. There was no statistical difference between ZTP group and control group at 1.5 h after modeling. Immunofluorescence results showed that the expression of P2X3 receptors in midbrain was positive in the rats of each group, mainly distributed in the nerve nuclei of periaqueductal gray (PAG), and the expression intencity of P2X3 receptors in migraine group was higher than control group and ZTP group. The protein positive levels of P2X3 receptors of migraine group and ZTP group were higher than control group (P<0.001), while the expression level of P2X3 receptor of ZTP group was lower than migraine group (P<0.001). Conclusion The effect of Zhengtian pills on the treatment of migraine may be related to its inhibition on the expression of P2X3 receptors in midbrain.