This study was performed to compare the effects of single and multiple irradiations of low-level laser therapy (LLLT) on bone regeneration in a mid-palatal suture following rapid palatal expansion (RPE). In this animal study, 40 male Wistar rats underwent RPE for 7 days and were divided into 4 groups including A: single LLLT on day 7, B: Multiple LLLT on days 7, 9, 11, 13 and 15, C: control (no LLLT), and D: sacrificed on day 7. Animals in group D were used to determine the amount of suture expansion. LLLT was done by a diode laser set at an 808 nm wavelength with a useful power output of 100 mW and duration of 0.1 ms. LLLT was applied to three points. After three weeks of retention, the rats were sacrificed and beheaded and the maxilla was evaluated by occlusal radiography, µ-CT, and histomorphometric analyses. A comparison of the mean measurements between the groups was performed using ANOVA and the Tukey post hoc test. Based on occlusal radiography and µCT, bone density in group B was significantly higher than group A and group C (<0.05). There was no significant difference in bone density between group A and group C (>0.05). Mean suture width (MSW) in group B was significantly lesser than the control group (=0.027) while there was no significant difference between MSWnin groups A and B (=0.116) and groups A and C (=0.317). It may be concluded that multiple low-power laser irradiation improves bone regeneration after RPE while single irradiation does not have a positive effect.