We cultured rat mesenchymal stem cells (MSCs) in a medium containing beta-glycerophosphate, ascorbic acid, and dexamethasone to show in vitro osteogenic differentiation of the MSCs. The differentiation was enhanced by adding solubilized type I collagen to the medium as evidenced by higher alkaline phosphatase activity as well as more calcium deposition than that without collagen. The exogenous collagen integrated well with the mineralized bone matrix and maintained the native triple helical structure. These findings indicate that exogenously supplemented type I collagen acts as a component of the extracellular matrix of MSCs, and deposited type I collagen facilitates osteogenic differentiation followed by maturation of mineralized bone matrix.