BACKGROUND: The hierarchical porous structure and surface topography of calcium phosphate (CaP) bioceramics have a crucial impact on their osteoinductivity. PURPOSE: To fabricate a biomimetic bone graft with an interconnected porous structure analogous to that of trabecular bone and a bioactive nanostructured surface with excellent osteoinductive potential. MATERIALS AND METHODS: A biphasic CaP (BCP) substrate with highly porous structure was fabricated by an improved sponge replication method. Surface modification was performed by uniformly depositing a hydroxyapatite (HA) nanoparticle layer to create nHA-coated BCP scaffolds. The effects of these scaffolds on osteogenic differentiation of murine bone marrow-derived stem cells (BMSCs) were investigated in vitro, and their osteoinductivity was further assessed in vivo. RESULTS: The BCP and nHA-coated BCP scaffolds had similar trabecular bone-like architectures but different surface structures, with mean grain sizes of ~55 nm and ~1 μm, respectively. Compared with the BCP substrate, the nHA-coated BCP scaffolds favored cell adhesion and promoted osteogenic differentiation of BMSCs, as evidenced by upregulated expression of osteogenic genes, enhanced alkaline phosphatase activity, and increased osteocalcin production. This could be attributed to activation of the BMP/Smad signaling pathway, as significantly higher expression levels of BMPRI, Smad1, Smad4, and Smad5 were observed in the nHA-coated BCP group. The nHA-coated BCP scaffold not only maintained scaffold integrity but also induced ectopic bone formation when implanted into rabbit dorsal muscle in vivo for 90 days, whereas the BCP substrate underwent marked biodegradation that led to severe inflammation with no sign of osteogenesis. CONCLUSION: The present study demonstrates the potential of this biomimetic bone graft with a trabecular framework and nanotopography for use in orthopedic applications.