Bone is a highly dynamic connective tissue that provides structural support, locomotion and acts as a shield for many vital organs from damage. Bone inherits the ability to heal after non-severe injury. In case of severe bone abnormalities due to trauma, infections, genetic disorders and tumors, there is a demand for a scaffold that can enhance bone formation and regenerate the lost bone tissue. In this study, a 3D collagen scaffold (CS) was functionalized and assessed under in vitro and in vivo conditions. For this, a collagen scaffold coated with hydroxyapatite (Ap-CS) was developed and loaded with a peptide LL-37. The physico-chemical characterisation confirmed the hydroxyapatite coating on the outer and inner surfaces of Ap-CS. In vitro studies confirmed that LL-37 loaded Ap-CS promotes osteogenic differentiation of human osteosarcoma cells without showing significant cytotoxicity. The efficacy of the LL-37 loaded Ap-CS for bone regeneration was evaluated at 4 and 12 weeks post-implantation by histopathological and micro-CT analysis in rabbit femur defect model. The implanted LL-37 loaded Ap-CS facilitated the new bone formation at 4 weeks compared with Ap-CS without LL-37. The LL-37 loaded Ap-CS incorporating apatite and peptide LL-37 would be useful as a multifunctional scaffold for bone tissue engineering.