Aim: Developing 3D scaffolds mimicking the nanoscale structure of the native extracellular matrix is important in tissue regeneration. In this study, we aimed to demonstrate the novelty of 3D nanofibrous scaffolds and compare their efficiency with 2D nanofibrous scaffolds. Materials & methods: The 2D poly(L-lactic acid)/collagen nanofibrous scaffolds were 2D meshes fabricated by the conventional electrospinning technique, whereas the 3D poly(L-lactic acid)/collagen nanofibrous scaffolds were fabricated by a modified electrospinning technique using a dynamic liquid support system. The morphology, proliferation and differentiation abilities of human mesenchymal stem cells in osteogenic medium on both scaffolds were investigated. Results & conclusion: Compared with the 2D scaffolds, the 3D scaffolds significantly increased the expression of osteoblastic genes of the stem cells as well as the formation of bone minerals. In addition, the scanning electron microscopic and micro-computed tomographic images showed the dense deposition of bone minerals aligned along the nanofibers of the 3D scaffolds after 14 and 28 days cultured with the mesenchymal stem cells. As such, the 3D electrospun poly(L-lactic acid)/collagen nanofibrous scaffold is a novel bone graft substitute for bone tissue regeneration. Original submitted 17 November 2011; Revised submitted 21 February 2012; Published online 18 June 2012