We have investigated the uptake of cationic polystyrene nanoparticles by mesenchymal stem cells (MSCs) using confocal fluorescence microscopy and flow cytometry. Two types of nanoparticles of about 100 nm diameter with similar zeta potentials were employed in this study, plain polystyrene (PS) nanoparticles and amino-functionalized polystyrene (NPS) nanoparticles, each carrying about 6000 amino groups on the surface. To assess the relative importance of specific endocytosis mechanisms, uptake was observed in the presence of the drugs dynasore and chlorpromazine. NPS nanoparticles were rapidly internalized and accumulated to a much higher level in MSCs than PS nanoparticles, predominantly via the main clathrin-mediated pathway. PS nanoparticles were internalized mainly via clathrin-independent endocytosis. The pronounced difference in the internalization behavior of PS and NPS nanoparticles points to specific interactions of the amino groups on the nanoparticle surface with the endocytosis machinery of the cells.