UNLABELLED The different transport pathways of 5-nm polymer-coated gold nanoparticles (Au NPs) crossing epithelial Caco-2 cell monolayers were explored. We found that the majority of cationic and neutral Au NPs depended heavily on endocytosis for cellular uptake and transport, and the anionic charged nanoparticles trafficked preferentially through the tight junctions (i.e., a paracellular pathway). The current study demonstrates that the surface chemistry of neutral polymer coatings dictate the trafficking through Caco-2 cell monolayers; poly(ethylene glycol)-coated Au NPs traffic via an endocytosis pathway assisted by microtubules; poly(2,3-hydroxy-propylacrylamide)-coated Au NPs traffic via endocytosis but assisted by other nonmicrotubular pathways. The Au NPs coated with poly(N-isopropylacrylamide) (hydrophobic above the lower critical solution temperature of 32°C) traffic via either the microtubule-assisted endocytosis pathway or the paracellular pathway depending on the temperature. This knowledge will aid in the future of the design of nanoparticles as potential oral drug carriers. FROM THE CLINICAL EDITOR The authors examined different transport pathways of polymer-coated gold nanoparticles to cross epithelial Caco-2 cells, concluding that surface chemistry of neutral polymer coatings dictates the trafficking through monolayers.