Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1α (SDF-1α)–directed chemotactic capacity to invade internal compartments of porous 3D bone substitute scaffolds in vitro and in vivo. In vitro native BMCSs invaded up to 500 μm into SDF-1α–releasing 3D scaffolds, whereas CXCR4-overexpressing BMSCs invaded up to 800 μm within 5 days. In addition, 60% downregulation of endogenous SDF-1 transcription in BMSCs by endoribonuclease-prepared siRNA before CXCR4 mRNA transfection enhanced SDF-1α–directed migration of human BMSCs by 50%. Implantation of SDF-1α–releasing scaffolds seeded with transiently CXCR4-overexpressing BMSCs resulted in an increase of invasion into internal compartments of the scaffolds in a mouse model. In vivo native BMCS invaded up to 250 μm into SDF-1α–releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 μm within 5 days. Thus, the SDF-1α/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1α–releasing 3D scaffolds in vitro and in vivo.