For oral vaccination, incorporation of antigens into nanoparticles has been shown to protect the antigen from degradation, but may also increase its uptake through the intestinal epithelium via M-cells. The aim of this study was to understand the mechanisms by which oral administration of antigen-loaded nanoparticles induces an immune response and to analyze the effect of the nanoparticle composition on these mechanisms. Nanoparticles made from chitosan (CS) and its N-trimethylated derivative, TMC, loaded with a model antigen ovalbumin (OVA) were prepared by ionic gelation with tripolyphosphate. Intraduodenal vaccination with OVA-loaded nanoparticles led to significantly higher antibody responses than immunization with OVA alone. TMC nanoparticles induced anti-OVA antibodies after only a priming dose. To explain these results, the interaction of nanoparticles with the intestinal epithelium was explored, in vitro, using a follicle associated epithelium model and visualized, ex vivo, using confocal laser scanning microscopy. The transport of FITC-OVA-loaded TMC nanoparticles by Caco-2 cells or follicle associated epithelium model was higher than FITC-OVA-loaded CS or PLGA nanoparticles. The association of nanoparticles with human monocyte derived dendritic cells and their effect on their maturation were determined with flow cytometry. TMC nanoparticles but not CS or PLGA nanoparticles had intrinsic adjuvant effect on DCs. In conclusion, depending on their composition, nanoparticles can increase the M-cell dependent uptake and enhance the association of the antigen with DC. In this respect, TMC nanoparticles are a promising strategy for oral vaccination.