Bone defects resulting from trauma or pathology represent a common and significant clinical problem. In this study, hydroxyapatite (HAp)-alumina bi-layered scaffolds, which have the benefits of both HAp (i.e., osteointegration, osteoconduction) and alumina (i.e., hardness) were used as a bone substitute for the repair of large segmental defects (20 mm) created in a beagle tibia model. Highly porous bi-layered scaffolds with isotropic-pore structures were fabricated using a polymer-template coating technique. The pore sizes obtained using this approach ranged between 230 μm and 470 μm, and porosity was 91.61±1.28%. Using scanning electron microscopy and energy dispersive spectroscopy, it was confirmed that the frame of each bi-layered scaffold consisted of an alumina inner layer and HAp outer layer. The evaluation of bone regeneration within each scaffold after implantation in the beagle tibia was performed using CT, micro-CT, scintigraphy. New bone formation was evident in the large segmental defects treated with HAp/alumina scaffolds. It was concluded from this study that the HAp/alumina bi-layered scaffold is instrumental in inducing host-scaffold engraftment at the distal and proximal ends of the defect as well as distributing the newly formed bone throughout each scaffold 8 weeks post-implantation.