The aim of this study was to determine the effectiveness of low-level laser therapy (LLLT) in the healing of experimentally created unicortical defect (UCD) of rat femurs. Twenty-four adult Wister rats were randomly divided into 2 groups: UCD and UCD+LLLT. The animals were subdivided into 3 groups (each group had 4 animals) and then they sacrificed at day 8,15 and 22 for radiological, pathological and biochemical analysis. Bone healing was significantly different between the groups at day 15 and 22 (P < 0.05), while bone formation was significantly increased in the UCD+LLLT group at day 22 compared to the UCD group (P < 0.05). On histopathological examination, significant difference was found between the UCD and UCD+LLLT groups on the 22nd day (P < 0.05) while there was no difference on the 8th and 15th day. In the UCD group, the plasma Alkaline phosphatase activity decreased on the 15th and 22nd days, Calcium on the 15th day and Phosphorus on the 8th and 22nd days compared to the UCD+LLLT group. In addition, Glutathione levels, Plasma Catalase, Glutathione Peroxidase and Superoxide Dismutase activity significantly reduced in the UCD group compared with UCD+LLLT on the 8th, 15th and 22nd day (P < 0.05). In conclusion, LLLT applied at a dose of 904 mu m accelerates fracture healing in UCD of a rat's femur. However, this effect shows an increase after the 22nd day of LLLT application.