The objective of this study was to assess the integrity and intestinal permeability of chitosan nanoparticles (CNPs) in simulated gastrointestinal fluids (SGIF). The cell-associated and transported chitosan was quantified and visualized after incubation of the CNPs with Caco-2 cell monolayer with/without SGIF treatment. In order to establish the role of proteins in the SGIF, CNPs were incubated with 4 proteins having different isoelectric points (pI). CNPs incubated with the fluids did not attach to the cell monolayer in contrast to the intact CNPs. Negatively-charged protein formed the complex with CNPs leading to particle size increase, but protected CNPs from disintegration. In contrary, positively-charged protein interacted with cross-linker causing disintegration of CNPs. CNPs incubated with the fluids did not attach to the cell monolayer in contrast to the intact CNPs. The results suggested that the surface charges of CNPs and proteins play a critical role in structural changes of CNPs in biological environment.