An in vitro cell culture system is developed for studying the uptake characteristics of nanoparticles (NPs) by endothelial cells under shear stress. Results show that the smaller polystyrene nanospheres are uptaken more than larger nanospheres for sizes ranging from 100 nm to 500 nm for 12, 24, and 48 hrs delivery times. While the result is similar to that found in static cultures, the observed trend is different from NP delivery behaviors to a simple glass surface in a flow, where no clear size dependence was observed because of repulsive electrostatic force on marginating NPs. The trend is also opposite to the behavior found in another study of the adhesion of labeled particles onto endothelial cells in whole blood flow. The comparison shows that the reduced zeta potential of NPs in a serum-containing cell medium and particle removal by cells results in reduced repulsive electrostatic force on marginating NPs. Consequently, the uptake behaviors are dominated by Brownian diffusion and cell membrane deformation process, which favor the uptake of NPs with reduced sizes.