Recently, the importance of nanofibers as a three-dimensional cell culture scaffold has been revealed. Research and development on nanofiber production using electrospinning has been actively conducted. Among various polymer polyvinyl alcohol (PVA), a water-soluble polyhydroxy polymer, is widely used due to its unique property of dissolving in a water, biocompatibility, biodegradability and permeability. However, PVA nanofiber is highly soluble in water which lead to low protein affinity and poor cell attachment. In this study, addition of PAA and GA to PVA solution resulted to the insolubility of the PVA nanofiber in water. RGD peptide, a major integrin binding domain, was mixed in PVA/PAA/GA solutions and fabricated using electrospinning. Cell adhesion and cell function were examined in RGD-PVA nanofiber membrane.. Surface morphology of nanofibers was analyzed by scanning electron microscope (SEM). MLE-12 cells and mouse hepatocytes were culture on PVA nanofibers and RGD-PVA nanofibers. The adherence, morphology, viability and proliferation of both cells were examined. As a result, cells cultured on RGD-PVA nanofibers showed better cell adhesion and growth stability then on PVA nanofibers alone. This study provides effective and simple way to confirm the possibility PVA nanofibers to be mixed with cell attaching peptides. Thus, RGD containing can be applied in three-dimensional cell culture.