The purpose of the present study was to evaluate the contributions of autogenous tooth bone graft (ATBG) combined with platelet-rich fibrin (PRF) on new bone formation and bone morphogenetic protein (BMP)-2 in rabbit calvarial defects. Twelve male New Zealand rabbits were used in this study. Three circular bone defects were prepared in each rabbit with a drill. These defects were divided into 3 groups: control, treated with ATBG, and treated with ATBG+PRF. The animals were sacrificed at 28 days. Samples were evaluated by histomorphometric analyses and total augmented area, new bone area and bone density were calculated. In addition, expression of BMP-2 was determined by immunohistochemical staining. The total augmented area, new bone area and bone density were significantly greater in the ATBG group than in the control group (P <0.05). Also, these values were significantly higher in the ATBG+PRF group than the ATBG group (P <0.05). Test groups demonstrated significantly increased BMP-2 levels compared with the control group (P <0.05). The present study suggested that ATBG combined with PRF significantly increased the new bone formation and enhanced bone healing in cranial defects.