In the design of proper scaffolds for tissue engineering, the bulk material properties and scaffold architecture are fundamental because the scaffolds have been shown to have an essential role in the induction of cell adhesion and mediation of cellular behavior. It is popularly accepted that the surface topography (shape, size, and surface texture) is one of the most vital cues that influences cellular reactions in vivo and cell-material interactions in vitro. The objective of this study was to better understand the impact of topography on MC3T3-E1 pre-osteoblasts' response to the co-culture of electrospun fibrous scaffolds. Three kinds of biomimetic scaffolds with typical average fiber diameters of 0.347 mu m, 0.947 mu m, and 6.48 mu m were fabricated by adjusting proper electrospinning conditions. Cell morphology, proliferation, ALP activity, and gene expression of MC3T3-E1 cells cultured on the scaffolds were studied. Cells cultured on 0.35 mu m scaffolds showed a higher projected area and upregulation of osteogenic phenotype gene, such as Runx2, Col I, ALP, and OCN. For cells cultured on 6.5 mu m scaffolds, a higher proliferation rate and aspect ratio were noted. Our results showed that topographical cues designed into scaffolds can regulate proliferation and differentiation of osteoblastic cells.