Artificial introduction of functional molecules on the cell surface may be a promising way to improve the therapeutic effects of cell therapy. Pegylated lipids are conventionally used in drug carriers. The lipid part of pegylated lipids noncovalently interacts with the cell surface. However, little information is available regarding conditions for cell-surface modification by using pegylated lipids. In this study, we synthesized fluorescein-labeled pegylated lipids and evaluated the factors that affect modification efficiency by using human mesenchymal stem cells (hMSCs). As the concentration of the pegylated lipid as well as the exposure time increased, the modification efficiency increased. The modification efficiency at 37°C was 20- and 3-fold higher than that at 4°C and 25°C, respectively. In addition, with an increase in the molecular weight of polyethylene glycol (PEG), more pegylated lipids were extracellularly distributed than those intracellularly distributed. At the optimal condition, pegylated lipids were observed mainly on the cell membrane by confocal microscopy. In contrast, the cell condition (adherent or nonadherent) had little or no effect on the cell-surface modification efficiency. The results of this study will be useful for constructing an optimal modification method for introducing functional molecules on the cell surface.