This study aimed to investigate and compare the effect of chitosan sponge and platelet-rich plasma (PRP) gel alone as well as their combination on bone regeneration in rabbit cranial defects. Four cranial defects with a 4.5-mm diameter were created in rabbit cranium and grafted with PRP, chitosan sponge alone, and chitosan sponge incorporated with PRP. The rabbits were killed by the fourth and eighth weeks, and the defects were analyzed histologically. Higher bone formation was observed in the PRP group when compared with the other groups at weeks 4 and 8. All parts of the defects were filled with thick trabecular new bone in the PRP group. The amount of new bone formation in the control groups was found to be less when compared with the PRP group and the least in the chitosan group. The defects that were filled with chitosan sponge showed a limited amount of new bone formation and an obvious fibrous demarcation line between chitosan particles and bone. Application of PRP showed a histological tendency toward increased bone formation. Other forms or derivatives of chitosan may have beneficial effects to achieve new bone regeneration.