A new approach for the preparation of hydrophilic chitosan nanoparticles (CS2NPs), based on an ionic gelation process, was established which was extremely mild and involved the mixture of two aqueous phases at room temperature. One phase contained polysaccharide chitosan (CS) and polyethylene oxide(PEO), and the other polyanion sodium tripolyphosphate (TPP). Size and zeta potential of the NPs could be modulated by varying the conditions for the preparation1 The new NPs prepared in our experiment showed great entrapment efficiency of insulin up to 90% and various factors had an influence on it. This hydrophilic system might be a very promising carrier for administration of therapeutic proteins and other macromolecules that were susceptible to interact with CS.