OBJECTIVE: To investigate the effects of estrogen on the threshold and temperature of orofacial pain and explore the influence on the function of glutamate and GABA neurons in the orofacial pain temperature perception pathway by observing the expression of vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter 1 (Vgat1). METHODS: A total of 24 adult female Sprague-Dawley rats were divided into three groups: sham operation (SHAM), ovariectomized (OVX) and ovariectomized plus estrogen intervention (OVX+E) (n = 8 per group). The threshold of mechanical pain of the orofacial region was assessed with von Frey filaments, and the temperature of the rat orofacial region was monitored by infrared thermography. Changes in the expression of Vglut2 and Vgat1 in glutamatergic and GABAergic neurons in the trigeminal ganglion (TG), spinal trigeminal nucleus (Sp5C), lateral parabrachial nucleus (LPB) and ventral posteromedial nucleus of the thalamus (VPM) were assessed by immunostaining and Western blotting. RESULTS: Under low-estrogen conditions, the mechanical pain threshold of the orofacial region of rats decreased significantly, and the temperature of the orofacial region increased significantly. The expression of Vglut2 and Vgat1 in the TG and Sp5C showed a downward trend, and the decline in Vgat1 was greater than that in Vglut2. Conversely, both proteins were upregulated in the LPB and VPM, and the magnitude of the changes in Vglut2 was greater than that in Vgat1. Estrogen therapy reversed these changes. CONCLUSION: Under low-estrogen conditions, the proportion of glutamate and GABA neurons in the orofacial pain and temperature sensation pathway changes, which leads to the imbalance of neurotransmission function and the enhancement of excitatory transmission of these two kinds of neurons and finally leads to a decrease in the orofacial pain threshold and an increase in temperature.