To treat large bone defects is a clinically challenging problem and utilizing tissue engineering technology is an attractive approach for overcoming such a problem. Previously, a biodegradable sponge incorporating bone morphogenic protein-2 (BMP-2), which can control the release of BMP-2 for a prolonged time in an in vivo environment, was reported. In addition, a biodegradable sponge composed of gelatin and β-tricalcium phosphate (βTCP), gelatin-βTCP sponge to develop a more ideal scaffold for enhancing bone regeneration was also created and previously reported. The purpose of this study was to investigate the effectiveness of the gelatin-βTCP sponge for the promotion of bone regeneration in a critical-sized bone defect site in vivo. Apparent bone regeneration was induced by the gelatin sponge incorporating BMP-2 and the gelatin-βTCP sponge with BMP-2 incorporation. In contrast, no apparent bone formation was induced by either the gelatin sponge only or the gelatin-βTCP sponge without BMP-2. To investigate the quality of the regenerated bone, we conducted a biomechanical evaluation with a three-point bending test. We found no significant difference between the gelatin sponge incorporating BMP-2 and the gelatin-βTCP sponge incorporating BMP-2 groups. Incorporation of βTCP into the gelatin sponge was expected to enhance biomechanical strength during the initial bone regeneration. However, our observations showed that the gelatin-βTCP sponge did not significantly improve the quality of regenerated bone from the viewpoint of biomechanical assessment, even though it did not impair the effectiveness of the promotion of bone regeneration by BMP-2 in the bone defect.