Although orthodontic treatment is common, orthodontic force often induced pain. Low-level laser therapy (LLLT) has been investigated to improve therapeutic comfort. In dentistry, LLLT is mainly applied using two types of lasers, CO 2 and diode lasers, whose biological actions are thought to be associated with wavelength (CO 2 : 10,600 nm; diode: 808 nm). The analgesic effect of LLLT on orthodontic treatment-related pain is widely reported but inconsistent. This study aimed to (1) determine whether irradiation with a CO 2 or diode laser attenuates orthodontic treatment-related pain using the jaw-opening reflex model, (2) elucidate the optimal irradiation protocol for both lasers to obtain the maximal analgesic effect, (3) evaluate the effects of laser irradiation on other biological features [e.g., tooth movement, glial fibrillary acidic protein (GFAP) expression, and temperature alterations] and (4) investigate the mechanism underlying the analgesic effect of laser irradiation. In this animal model, orthodontic treatment-induced pain manifested as a significantly reduced the threshold for inducing the jaw-opening reflex on the orthodontically treated side compared with the contralateral side. GFAP expression in the bilateral trigeminal ganglia (TGs) was significantly increased by the application of orthodontic force. CO 2 laser irradiation of the orthodontically treated region significantly increased the threshold for inducing the jaw-opening reflex and the peripheral temperature. Similar reductions in jaw-opening reflex excitability were induced by surface anesthesia and thermal stimulation but not, the diode laser. Neither CO 2 nor diode laser irradiation altered GFAP expression in the TGs. Infiltration anesthesia also significantly increased the threshold for inducing the jaw-opening reflex on each anesthetized side. Irradiation (30 s) by either laser immediately after orthodontic force application (preirradiation) significantly decreased jaw-opening reflex excitability and GFAP expression in the bilateral TGs the next day. However, thermal stimulation immediately after orthodontic force application failed to alter jaw-opening reflex excitability the next day. Laser irradiation did not alter tooth movement; however, Tsuchiya et al.Beneficial Effects of CO 2 Laser an optimized irradiation protocol for aiding tooth movement is suggested. In conclusion, both CO 2 and diode lasers are able to prevent orthodontic treatment-related pain. Furthermore, the involvement of temperature alterations and surface anesthesia in the analgesic effect induced by CO 2 laser irradiation is suggested.