Tenascin C (TNC) is an extracellular matrix glycoprotein involved in osteogenesis and bone mineralization. In a previous study, we identified TNC protein located in the matrix vesicles (MVs) of osteoblasts. MVs are determinant in the mineralization formation. Therefore, we hypothesize whether TNC can modulate osteoblast mineralization via MVs. In this study, we demonstrated that the expression level of TNC was increased with osteoblast differentiation of osteoblast-like SaOS2 cells, and down-regulation of TNC expression by siRNA could significantly inhibit SaOS2 differentiation toward osteoblasts and mineralization as evidenced by decreases in ALP activity, mineralized nodule formation, calcium deposition, and down-regulation of osteogenic marker genes ALP, and COL1A1. Furthermore, we validated that TNC located in the MVs of mineralized SaOS2 cells, and that down-regulation of TNC could decrease MVs mineralization ability in vitro, and the decrease of MVs mineralization ability was not associated with annexins. In conclusion, in this study, we extended the role of TNC during osteogenesis previous progresses, and that supported TNC as an important functional MVs component in modulating osteoblast mineralization.