Bone is known to have a natural function to heal itself. However, if the bone damage is beyond a critical degree, intervention such as bone grafting may be imperative. In this work, the fabrication of a novel bone scaffold composed of natural bone components and polycaprolactone (PCL) using 3D printing is put forward. α1, 3-galactosyltransferase deficient pigs were used as the donor source of a xenograft. Decellularized porcine bone (DCB) with attenuated immunogenicity was used as the natural component of the scaffold with the aim to promote bone regeneration. The 3D printed DCB-PCL scaffolds com-bined essential advantages such as uniformity of the interconnected macropores and high porosity and enhanced compressive strength. The biological properties of the DCB-PCL scaffolds were evaluated by studying cell adhesion, viability, alkaline phosphatase activity and osteogenic gene expression of human bone marrow-derived mesenchymal stem cells. The in vitro results demonstrated that the DCB-PCL scaffolds exhibit an enhanced performance in promoting bone differentiation, which is correlated to the DCB content. Furthermore, critical-sized cranial rat defects were used to assess the effect of DCB-PCL scaffolds on bone regeneration in vivo. The results confirm that in comparison with PCL scaffolds, the DCB-PCL scaffolds can significantly improve new bone formation in cranial defects. Thus, the proposed 3D printed DCB-PCL scaffolds emerge as a promising regeneration altative in the clinical treatment of large bone defects.