Self-assembled nanoparticles (NPs) from arginine-modified chitosan (CS-N-Arg) and thiolated fucoidan (THL-fucoidan) were synthesized to enhance the transport of dextran and curcumin across intestinal epithelial cell layer. CS-N-Arg/THL-fucoidan NPs exhibited a pH-sensitive assembly-disassembly and drug release property. Evaluations of the NPs in enhancing the transport of a hydrophilic macromolecule (FITC-dextran) and a hydrophobic drug (curcumin) were investigated in Caco-2 cell monolayers. The cationic CS-N-Arg in the NPs induced disruption of intestinal epithelial tight junctions as indicated by the decrease of transepithelial electrical resistance (TEER). Permeation studies revealed that the NPs enhanced the paracellular permeation of macromolecular dextran through the monolayer barrier. In addition, the multifunctional NPs increased the permeability of rhodamine 123 because the thiomer THL-fucoidan in the NPs inhibited P-glycoprotein. Cellular uptake and permeability of curcumin encapsulated in the NPs were improved due to increasing their water solubility and stability.