Recruitment of mesenchymal stem cells (MSCs) to an injury site and their differentiation into the desired cell lineage are implicated in deficient bone regeneration. To date, there is no ideal structure that provides these conditions for bone regeneration. In the current study, we aim to develop a novel scaffold that induces MSC migration towards the defect site, followed by their differentiation into an osteogenic lineage. We have fabricated a gelatin/nano-hydroxyapatite (G/nHAp) scaffold that delivered cannabidiol (CBD)-loaded poly (lactic-co-glycolic acid) (PLGA) microspheres to critical size radial bone defects in a rat model. The fabricated scaffolds were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), and then analyzed for porosity and degradation rate. The release profile of CBD from the PLGA microsphere and CBD-PLGA-G/nHAp scaffold was analyzed by fluorescence spectroscopy. We performed an in vitro assessment of the effects of CBD on cellular behaviors of viability and osteogenic differentiation. Radiological evaluation, histomorphometry, and immunohistochemistry (IHC) analysis of all defects in the scaffold and control groups were conducted following transplantation into the radial bone defects. An in vitro migration assay showed that CBD considerably increased MSCs migration. qRT-PCR results showed upregulated expression of osteogenic markers in the presence of CBD. Histological and immunohistochemical findings confirmed new bone formation and reconstruction of the defect at 4 and 12 week post-surgery (WPS) in the CBD-PLGA-G/nHAp group. Immunofluorescent analysis revealed enhanced migration of MSCs into the defect areas in the CBD-PLGA-G/nHAp group in vivo. Based on the results of the current study, we concluded that CBD improved bone healing and showed a critical role for MSC migration in the bone regeneration process.