Orthodontic pain that is induced by tooth movement is an important sequela of orthodontic treatment and has a significant effect on patient quality of life. Studies have shown that the high expression of transient receptor potential vanilloid 1 (TRPV1) in trigeminal ganglions plays a vital role in the transmission and modulation of orofacial pain. However, little is known about the role of TRPV1 in orthodontic pain. In this study, male Sprague–Dawley rats were randomly assigned to six groups to study the role of TRPV1 in the modulation of tooth-movement pain. The expression levels of TRPV1 mRNA and protein were determined by real-time PCR and western blot, respectively. Moreover, pain levels were assessed using the rat grimace scale (RGS). The role of TRPV1 in modulating tooth-movement pain was examined by injecting a TRPV1 antagonist into the trigeminal ganglia of rats. A lentivirus containing a TRPV1 shRNA sequence was constructed and transduced into the rats’ trigeminal ganglia. The results showed that the expression levels of TRPV1 protein and mRNA were elevated following tooth-movement pain. Pain levels increased rapidly on the 1
st
day, peaked on the 3
rd
day and returned to baseline on the 14
th
day. The TRPV1 antagonist significantly reduced tooth-movement pain. The lentivirus containing a TRPV1 shRNA sequence was able to inhibit the expression of TRPV1 and relieved tooth-movement pain. In conclusion, TRPV1-based gene therapy may be a treatment strategy for the relief of orthodontic pain.