Targeted drug delivery is an important research area in specific therapy. Transferrin-conjugated nanoparticles are an attractive formulation as a vehicle for specific cellular uptake and targeted drug delivery. In this report, atomic force microscopy imaging was used to visualize the process of cellular uptake of transferrin-coupled gold nanoparticles on the surfaces of live cells for the first time. High-resolution images were captured, showing the endocytosis of transferrin-conjugated nanoparticles taking place during the process of internalization. This specific transferrin-mediated nanoparticle uptake was validated by confocal scanning imaging and transferrin competition experiments.