Many synthetic bone materials have been studied for their potential of regenerative effects in periodontal tissue. Safflower seeds have been traditionally used as a drug for the treatment of fracture and blood stasis in oriental medicines. The purpose of this study was to assess and compare the osseous responses in rat calvarial defects between bone substitutes such as calcium carbonate and bovine-derived hydroxyapatite and feeding of safflower seeds. The calvarial defects were made with 8 mm trephine bur in 24 Sprague-Dawley rats. Two graft materials were implanted in each experimental groups, whereas the control and safflower seed feeding groups were sutured without any other treatment. And then the rats of safflower seed feeding group were supplied with 3 g/day of safflower seeds. Each group was sacrificed at 4 weeks and 8 weeks. To study a histopathology related to bone healing and regeneration, Goldner's Masson Trichrome stain was done at each weeks. The tissue response was evaluated under light microscope. There were more osteoblastic activity, new bone formation, dense bony connective tissues in bovine-derived hydroxyapatite group compared to other groups at 8 weeks. The osseous defect area of safflower seed feeding group was filled with prominent fibrous tissues, where less inflammatory infiltration and new capillary proliferation. In the early phase of bone healing, safflower seed feeding reduces the inflammatory response and promotes the proliferation of connective tissue. These results suggest that natural bovine-derived HA and safflower seed feeding could enhance the regenerative potential in periodontal defects.