Manufactured nanomaterials provide promising features for new technologies in cosmetic, food, and pharmaceutical applications. On the other hand, orally ingested nanomaterials/ nanoparticles may interact with or enter intestinal cells via different mechanisms, resulting in possible injuries of the biological system. For that reason, the current study aims to provide useful information concerning physicochemical properties of nanoparticles with regard to cytotoxic effects and uptake mechanisms in the small intestine. Differently charged polystyrene nanoparticles were used and cytotoxicity and uptake were studied with an intestinal in vitro co-culture model, mimicking the villus epithelium and a triple-culture model recapitulating the follicle-associated epithelium. Mechanisms of cellular transport were investigated at 37°C and 4°C to verify that internalization mainly occurs energy-dependently. Chemical inhibitors (i.e., chlorpromazine, genistein, dynasore) were used to block dynamin-dependent endocytic pathways without affecting cell viability and membrane integrity. Qualification and quantification were performed via confocal microscopy and flow cytometry. Furthermore, co-localization studies with commonly used markers (i.e., transferrin, lactosylceramide) were carried out and co-localization was assessed via calculation of Pearson´s correlation coefficient and Mander´s overlap coefficient. The results show that size and surface chemistry play a crucial role in cytotoxic interactions and cellular uptake of nanoparticles (NPs). Independent of the surface charge, NPs strongly interact with intestinal mucus and are immobilized. Uptake predominantly occurs via M cells and is surface-charge dependent. Whereas negatively charged particles fail to enter cells, positive and neutral particles penetrate M cells energy-dependently. More precisely, both clathrin- and caveolae-mediated endocytosis are involved. It can be concluded that the presented system serves as a valuable tool to assess safety aspects of manufactured nanomaterials and hence, substantially contributes to nanosafety efforts.