The behavior of human osteoblast-like MG63 cells cultured on electrospun collagen fibers of three different sizes (50-200 nm, 200-500 nm, and 500-1000 nm in diameter) were investigated. The growth of MG63 cells on all three electrospun collagen fibers are the same and about 70% higher than those cultured on monomeric collagen and tissue-culture polystyrene (TCPS). The migration speed of MG63 cells, on the other hand, decreased as the diameter of nanofibers increased. There were more distinct actin stress fibers formed in MG63 cells when the cells cultured on collagen substrates as compared with TCPS. In addition, MG63 cells displayed different adhesion and spreading patterns on different sizes of collagen fibers. Size variation of collagen nanofibers apparently has more impact on cell migration distance and cell morphology as compared with cell growth. It was demonstrated that collagen nanofibers promoted MG63 cell interaction with matrices by providing a suitably rough nanometer surface. The results of this study present important information for the development of collagen-based biomaterials.