As most of polypeptides are marginally stable, a mild formulation procedure would be beneficial for the activities of these drugs. The objective of the present study was to develop a novel pH-sensitive nanoparticle system that was suitable for entrapment of hydrophilic insulin but without affecting its conformation. Chitosan was incorporated as a positively charged material, and one of the three poly(methylmethacrylate/methylmethacrylic acid) copolymers, consisting of Eudragit L100-55, L100, and S100, was used as a negatively charged polymer for preparation of three insulin nanoparticles, respectively. Three nanoparticles obtained were spherical. The mean diameters were in the range from 200 nm to 250 nm, and the entrapment efficiencies, from 50% to 70%. The surface analysis indicated that insulin was evenly distributed in the nanoparticles. Polymer ratio of chitosan to Eudragit was the factor which influenced the nanoparticles significantly. Characterization results showed that the electrostatic interactions existed, thus providing a mild formulation procedure which did not affect the chemical integrity and the conformation of insulin. In vitro release studies revealed that all three types of the nanoparticles exhibited a pH-dependant characteristic. The modeling data indicated that the release kinetics of insulin was nonlinear, and during the release process, the nanoparticles showed a polynomial swelling. On overall estimation, the insulin chitosan-Eudragit L100-55 nanoparticles may be better for the oral delivery. This new pH-sensitive nanoparticle formulation using chitosan and Eudragit L100-55 polymer may provide a useful approach for entrapment of hydrophilic polypeptides without affecting their conformation.