Nanomaterials can mimic properties of extracellular matrix molecules, promising great potential for scaffold composition in tissue engineering. In the present study, we investigated whether barium titanate nanoparticles (BT NP) combined with alginate polymer would provide a new cytocompatible three-dimensional (3D) scaffold to induce osteogenic stem cell differentiation. In vitro cytocompatibility and osteogenic differentiation potential were investigated using human mesenchymal stem cells (MSC). Firstly, we studied the cell viability and oxidative stress by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) thiazolyl blue tetrazolium bromide (MTT) and superoxide dismutase (SOD) assays. Overall, neither pure BT NP or BT NP/alginate 3D scaffold induced cytotoxicity. The scanning electron and atomic force microscopy revealed that BT NP/alginate 3D scaffold produced exhibited highly interconnected pores and surface nanotopography that were favorable for MSC differentiation. Von Kossa staining showed mineralization nodules and MSCs morphology changed from spindle to cuboid shape after 21 d. Finally, BMP-2 and ALP mRNA were significantly upregulated on cells grown into the BT NP/alginate 3D scaffold. Thus, the BT NP/alginate 3D scaffold showed an osteogenic differentiation induction potential, without the addition of osteogenic supplements. These results indicate that the BT NP/alginate 3D scaffold provides a cytocompatible and bioactive microenvironment for osteogenic human MSC differentiation.