Purpose In clinical use of low-level laser therapy for bone regeneration (LLLT), application protocol (dose, duration, and repetitions) has not been established. This study aimed to depict a reliable dosage of LLLT by evaluating the efficacy of different dosing of LLLT (diode) on the healing of rabbit cranial defects. Methods Critical size defects were prepared in calvarias of 26 New Zealand White Rabbits in such each animal containing both test and control groups. Test groups were irradiated with 4 Joule/cm(2) (j/cm(2)), 6 j/cm(2), and 8 j/cm(2). The rabbits were subjected to six times of laser treatments in 10 days. At the end of the second week, 5 rabbits were sacrificed for histopathological and immunohistochemical analyses. At the 4th and 8th weeks, 20 rabbits (10 each) were sacrificed for micro-CT and histopathological analyses. Results Micro-CT evaluation revealed improved new bone formation in all test groups compared to the control group. 6 j/cm(2) group demonstrated the highest bone formation. The highest bone morphogenic protein -2 levels were found in the 4 j/cm(2) group. Osteocalcin expression was significantly higher in 4 j/cm(2) group. Conclusions Our findings indicate that LLLT have a positive effect on new bone formation. The high efficacy of doses of 4 j/cm(2) and 6 j/cm(2) is promising to promote early bone healing.