Objective To investigate the effect of hyperbaric oxygen (HBO) combined with basic fibroblast growth factor (bFGF) on bone defect repair with natural calcined bovine bone (CBB), and to explore its relationship with the expression of osteoprotegerin (OPG) and CD34. Methods A total of 32 SD rats were randomly divided into two groups:HBO and non-HBO (NHBO) groups (n=16 in each group). Bone defects were made on the left and right sides of the top of the skull. CBB (CBB group) or CBB combined with bFGF (CBBbF group) were implanted on one side and non-implanted on the other side (control[Con] group). HBO group received HBO exposure for 2 weeks, and the rats were sacrificed at 4, 8 and 12 weeks after operation. The healing of bone defect was observed by radiographic and histological observation, and the expression of OPG and CD34 was evaluated by immunohistochemistry. Results The bone densities (BDs) of bone defect areas of the rats were significantly higher in the HBO-CBB, HBO-CBBbF, NHBO-CBB and NHBO-CBBbF groups than in the HBO-Con and NHBO-Con groups at 12 weeks after operation, and the HBO-CBBbF group has the highest BD (all P<0.01). The percentages of new bone formation areas were significantly higher in the HBO-CBB, HBO-CBBbF, NHBO-CBB and NHBO-CBBbF groups than in the HBO-Con and NHBO-Con groups (all P<0.01), and the blood vessel numbers were significantly larger in the HBO-CBBbF, HBO-CBB, NHBO-CBB and NHBO-CBBbF groups than in the HBO-Con and NHBO-Con groups at 12 weeks after operation. The blood vessel numbers were significantly larger in the HBO-CBBbF, HBO-CBB and NHBO-CBBbF groups than in the NHBO-CBB group at each time point after operation. The expression of OPG and CD34 was significantly higher in the HBO-CBBbF, HBO-CBB and NHBO-CBBbF groups than in the NHBO-CBB, HBO-Con and NHBO-Con groups (P<0.05, P<0.01). Conclusion CBB can promote bone defect repair and bFGF can increase bone regeneration and blood vessel number. HBO has synergistic effect on bFGF combined with CBB in repairing bone defects, and the effect is closely related to the promotion of OPG and CD34 expression.