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, CO2 and diode lasers, whose biological actions are thought to be associated with wavelength (CO2: 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 CO2 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. CO2 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 CO2 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, an optimized irradiation protocol for aiding tooth movement is suggested. In conclusion, both CO2 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 CO2 laser irradiation is suggested.